Host cells for use in an inducible coexpression system

ABSTRACT

The present invention provides host cells for use in an inducible coexpression system that is capable of controlled induction of expression of each gene product.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/419,653, which is a national stage entry under 35 U.S.C. §371 ofInternational Application No. PCT/US2013/053562 filed 5 Aug. 2013, whichclaims the benefit of U.S. Provisional Application No. 61/679,751, filedon 5 Aug. 2012, and U.S. Provisional Application No. 61/747,246, filedon 29 Dec. 2012, the entire disclosures of which are incorporated byreference herein.

REFERENCE TO THE SEQUENCE LISTING

This application includes a sequence listing submitted electronically,in a file entitled “AbSci-001PCTUS-ST25”, created on 4 Feb. 2015 andhaving a size of 137 kilobytes (KB), which is incorporated by referenceherein.

FIELD OF THE INVENTION

The present invention is in the general technical fields of molecularbiology and biotechnological manufacturing. More particularly, thepresent invention is in the technical field of recombinant proteinexpression.

BACKGROUND OF THE INVENTION

Production of biotechnological substances is a complex process, evenmore so when the desired product is a combination of molecules encodedby different genes, such as a multimeric protein formed from two or moredifferent polypeptides. Successful coexpression of multiple geneproducts requires overcoming a number of challenges, which arecompounded by the simultaneous expression of more than one gene product.Problems that must be overcome include creating compatible expressionvectors when more than one type of vector is used; obtaining the correctstoichiometric ratio of products; producing gene products that arefolded correctly and in the proper conformation with respect to bindingpartners; purifying the desired products away from cells and unwantedproteins, such as proteins that are folded incorrectly and/or are in anincorrect conformation; and minimizing the formation of inclusionbodies, as one aspect of maximizing the yield of the desired product(s).Many different approaches have been taken to address these challenges,but there is still a need for better coexpression methods.

Several inducible bacterial protein expression systems, includingplasmids containing the lac and am promoters, have been devised toexpress individual proteins. These systems have limited utility in thecoexpression of difficult-to-express proteins as they fail to induceprotein homogenously within the entire growth culture population inwild-type E. coli (Khlebnikov and Keasling, “Effect of lacY expressionon homogeneity of induction from the P_(tac) and P_(trc) promoters bynatural and synthetic inducers”, Biotechnol Prog 2002 May-June; 18(3):672-674). When expression of the transport proteins for inducers isdependent on the presence of inducer, as is the case for wild-type E.coli lac and am systems, the cellular concentration of the inducer mustreach a threshold level to initiate the production of transportproteins, but once that threshold has been reached, an uncontrolledpositive feedback loop can occur, with the result being a high level ofinducer in the cell and correspondingly high levels of expression frominducible promoters: the “all-or-none” phenomenon. Increasing theconcentration of the inducer in the growth medium increases theproportion of cells in the population that are in high-expression mode.Although this type of system results in concentration-dependentinduction of protein expression at the population scale, it issuboptimal for expression and production of proteins that require tightcontrol of expression, including those that are toxic, have poorsolubility, or require specific concentrations for other reasons.

Some efforts have been made to address the “all-or-none” inductionphenomenon in single-promoter expression systems, by eliminatinginducer-dependent transport of the inducer. One example is having a nullmutation in the lactose permease gene (lacYam) and using an alternateinducer of the lac promoter such as IPTG(isopropyl-thio-β-D-galactoside), which can get through the cellmembrane to some degree in the absence of a transporter (Jensen et al.,“The use of lac-type promoters in control analysis”, Eur J Biochem 1993Jan. 15; 211(1-2): 181-191). Another approach is the use of anarabinose-inducible promoter in a strain deficient in the arabinosetransporter genes, but with a mutation in the lactose permease gene,lacY(A117C), that allows it to transport arabinose into the cell(Morgan-Kiss et al., “Long-term and homogeneous regulation of theEscherichia coli araBAD promoter by use of a lactose transporter ofrelaxed specificity”, Proc Natl Acad Sci USA 2002 May 28; 99(11):7373-7377).

The components of individual protein expression systems are oftenincompatible, precluding their use in coexpression systems, as they maybe adversely affected by ‘crosstalk’ effects between differentinducer-promoter systems, or require mutually exclusive genomicmodifications, or be subject to general metabolic regulation. An attemptto address the ‘crosstalk’ problem between the lac and am induciblepromoter systems included directed evolution of the AraC transcriptionalactivator to improve its ability to induce the araBAD promoter in thepresence of IPTG, an inducer of the lac promoter (Lee et al., “Directedevolution of AraC for improved compatibility of arabinose- andlactose-inducible promoters”, Appl Environ Microbiol 2007 September;73(18): 5711-5715; Epub 2007 Jul. 20). However, the compatibilitybetween expression vectors based on ara and lac inducible promoters isstill limited due to the requirement for mutually exclusive genomicmodifications: a lacY point mutation (lacY(A117C)) for homogenousinduction of the araBAD promoter by arabinose, and a null lacY gene forhomogenous induction of the lac promoter by IPTG. General metabolicregulation—for example, carbon catabolite repression (CCR)—can alsoaffect the compatibility of inducible promoters. CCR is characterized bythe repression of genes needed for utilization of a carbon-containingcompound when a more preferred compound is present, as seen in thepreferential use of glucose before other sugars. In the case of the araand prp inducible promoter systems, the presence of arabinose reducesthe ability of propionate to induce expression from the prpBCDEpromoter, an effect believed to involve CCR (Park et al., “The mechanismof sugar-mediated catabolite repression of the propionate catabolicgenes in Escherichia coli”, Gene 2012 Aug. 1; 504(1): 116-121, Epub 2012May 3). There is clearly a need for an inducible coexpression systemthat overcomes these problems.

SUMMARY OF THE INVENTION

The present invention provides inducible coexpression systems capable ofcontrolled-induction of each gene product component.

One embodiment of the invention is a host cell comprising two or moretypes of expression constructs, wherein the expression construct of eachtype comprises an inducible promoter and a polynucleotide sequenceencoding a gene product, said polynucleotide sequence to be transcribedfrom the inducible promoter; wherein (1) at least one of said induciblepromoters is responsive to an inducer that is not an inducer of anotherof said inducible promoters; and at least one of said gene productsforms a multimer with another of said gene products; or (2) at least oneof said gene products is selected from the group consisting of: (a) apolypeptide that lacks a signal peptide and that forms at least threedisulfide bonds; (b) a polypeptide selected from the group consisting ofarabinose- and xylose-utilization enzymes; and (c) a polypeptideselected from the group consisting of lignin-degrading peroxidases.Another embodiment of the invention is a host cell comprising two ormore types of expression constructs, wherein the expression construct ofeach type comprises an inducible promoter and a polynucleotide sequenceencoding a gene product, said polynucleotide sequence to be transcribedfrom the inducible promoter; wherein each inducible promoter is not alactose-inducible promoter; and wherein at least one of said geneproducts is a polypeptide that forms at least two disulfide bonds, orforms at least two and fewer than seventeen disulfide bonds, or forms atleast two and fewer than ten disulfide bonds, or forms a number ofdisulfide bonds selected from the group consisting of two, three, four,five, six, seven, eight, and nine. In some embodiments of the invention,this host cell is a prokaryotic cell, and in some instances, it is an E.coli cell. In other embodiments of the invention, the host cell is aeukaryotic cell, and in some instances it is a yeast cell, and in somefurther instances it is a Saccharomyces cerevisiae cell. In furtherembodiments, the expression constructs comprised by a host cell eachcomprise at least one inducible promoter, wherein the inducible promoteris an L-arabinose-inducible promoter or a propionate-inducible promoter,or is selected from the group consisting of: the araBAD promoter, theprpBCDE promoter, the rhaSR promoter, and the xlyA promoter, or whereinthe inducible promoter is not a lactose-inducible promoter. Inadditional embodiments, at least one expression construct comprised by ahost cell further comprises a polynucleotide sequence encoding atranscriptional regulator that binds to an inducible promoter; in someembodiments, the polynucleotide sequence encoding a transcriptionalregulator and the inducible promoter to which said transcriptionalregulator binds are in the same expression construct; and in furtherinstances, the transcriptional regulator is selected from the groupconsisting of: AraC, PrpR, RhaR, and XylR; or in particular is AraC, orPrpR. In certain embodiments, at least one expression constructcomprised by a host cell was produced by a method comprising a step ofinserting a polynucleotide sequence into a plasmid selected from thegroup consisting of: pBAD18, pBAD18-Cm, pBAD18-Kan, pBAD24, pBAD28,pBAD30, pBAD33, pPRO18, pPRO18-Cm, pPRO18-Kan, pPRO24, pPRO30, andpPRO33; or particularly into pBAD24 or pPRO33. Other examples of theinvention include a host cell comprising two or more types of expressionconstructs, wherein the expression construct of each type comprises aninducible promoter and a polynucleotide sequence encoding a geneproduct, wherein at least one gene product is a polypeptide, or isselected from the group consisting of: (a) an immunoglobulin heavychain; (b) an immunoglobulin light chain; and (c) a fragment of any of(a)-(b), or is an immunoglobulin light chain, or is an immunoglobulinheavy chain; or is an infliximab heavy or light chain or a fragmentthereof, or has at least 80% or 90% amino acid sequence identity withSEQ ID NO:30 or SEQ ID NO:31 across at least 50% or 80% of the length ofSEQ ID NO:30 or SEQ ID NO:31, respectively, or has the amino acidsequence of SEQ ID NO:30 or SEQ ID NO:31.

In additional embodiments of the invention, the host cell comprises twoor more types of expression constructs, wherein the expression constructof each type comprises an inducible promoter and a polynucleotidesequence encoding a gene product, said polynucleotide sequence to betranscribed from the inducible promoter; wherein at least one geneproduct is a polypeptide that lacks a signal peptide and that forms atleast three disulfide bonds, or at least three and fewer than seventeendisulfide bonds, or at least eighteen and fewer than one hundreddisulfide bonds, or at least three and fewer than ten disulfide bonds,or at least three and fewer than eight disulfide bonds; or forms anumber of disulfide bonds selected from the group consisting of three,four, five, six, seven, eight, and nine; or is a polypeptide selectedfrom the group consisting of: (a) an immunoglobulin heavy chain; (b) animmunoglobulin light chain; (c) manganese peroxidase; and (d) a fragmentof any of (a)-(c); or is an infliximab heavy or light chain or afragment thereof, or has at least 80% or 90% amino acid sequenceidentity with SEQ ID NO:30 or SEQ ID NO:31 across at least 50% or 80% ofthe length of SEQ ID NO:30 or SEQ ID NO:31, respectively, or has theamino acid sequence of SEQ ID NO:30 or SEQ ID NO:31; or has at least 80%or 90% amino acid sequence identity with SEQ ID NO:13, SEQ ID NO:15, orSEQ ID NO:23 across at least 50% or 80% of the length of SEQ ID NO:13,SEQ ID NO:15, or SEQ ID NO:23, respectively, or has the amino acidsequence of SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:23; or is apolypeptide selected from the group consisting of arabinose- andxylose-utilization enzymes such as xylose isomerase; or is a polypeptideselected from the group consisting of lignin-degrading peroxidases, suchas manganese peroxidase or versatile peroxidase.

In further embodiments of the invention, a host cell is provided whichcomprises two types of expression constructs, and in certain instances,one type of expression construct is produced by a method comprising astep of inserting a polynucleotide sequence into a pBAD24 polynucleotidesequence, and the other type of expression construct is produced by amethod comprising a step of inserting a polynucleotide sequence into apPRO33 polynucleotide sequence.

Another instance of the invention is a host cell comprising two or moretypes of expression constructs, wherein the expression construct of eachtype comprises an inducible promoter, and wherein the host cell has analteration of gene function of at least one gene encoding a transporterprotein for an inducer of at least one said inducible promoter, and asanother example, wherein the gene encoding the transporter protein isselected from the group consisting of araE, araF, araG, araH, rhaT,xylF, xylG, and xylH, or particularly is araE. As a further embodiment,a host cell is provided comprising two or more types of expressionconstructs, wherein the expression construct of each type comprises aninducible promoter, and wherein the host cell has a reduced level ofgene function of at least one gene encoding a protein that metabolizesan inducer of at least one said inducible promoter, and as furtherexamples, wherein the gene encoding a protein that metabolizes aninducer of at least one said inducible promoter is selected from thegroup consisting of araA, araB, araD, prpB, prpD, rhaA, rhaB, rhaD,xylA, and xylB. As an additional example, a host cell is providedcomprising two or more types of expression constructs, wherein theexpression construct of each type comprises an inducible promoter, andwherein the host cell has a reduced level of gene function of at leastone gene encoding a protein involved in biosynthesis of an inducer of atleast one said inducible promoter, which in further embodiments isselected from the group consisting of scpA/sbm, argK/ygfD, scpB/ygfG,scpC/ygfH, rmlA, rmlB, rmlC, and rmlD.

The invention also provides a host cell comprising two or more types ofexpression constructs, wherein the expression construct of each typecomprises an inducible promoter, and wherein the host cell has analtered gene function of a gene that affects the reduction/oxidationenvironment of the host cell cytoplasm, which in some examples isselected from the group consisting of gor and gshB; or wherein the hostcell has a reduced level of gene function of a gene that encodes areductase, which in some embodiments is trxB; or wherein the host cellcomprises at least one expression construct encoding at least onedisulfide bond isomerase protein, which in some embodiments is DsbC; orwherein the host cell comprises at least one polynucleotide encoding aform of DsbC lacking a signal peptide; or wherein the host cellcomprises at least one polynucleotide encoding Erv1p.

In other aspects of the invention, a host cell is provided comprisingtwo or more types of expression constructs, wherein the expressionconstruct of each type comprises an inducible promoter, wherein theexpression construct of each type comprises an inducible promoter thatis not an inducible promoter of the expression construct of each othertype, or wherein the expression construct of each type comprises anorigin of replication that is different from the origin of replicationof the expression construct of each other type.

As a particular example of the invention, an E. coli host cell isprovided, comprising two types of expression constructs, wherein onetype of expression construct is produced by a method comprising a stepof inserting a polynucleotide sequence into a pBAD24 polynucleotidesequence, and the other type of expression construct is produced by amethod comprising a step of inserting a polynucleotide sequence into apPRO33 polynucleotide sequence; and the host cell further comprising twoor more of the following: (a) a deletion of the araBAD genes; (b) analtered gene function of the araE and araFGH genes; (c) a lacY(A177C)gene; (d) a reduced gene function of the prpB and prpD genes; (e) areduced gene function of the sbm/scpA-ygfD/argK-ygfGH/scpBC genes,without altering expression of the ygfI gene; (f) a reduced genefunction of the gor and trxB genes; (g) a reduced gene function of theAscG gene; (h) a polynucleotide encoding a form of DsbC lacking a signalpeptide; and (i) a polynucleotide encoding Erv1p, ChuA, or a chaperone;and in certain examples, the host cell further comprises at least oneexpression construct comprising a polynucleotide sequence encoding agene product, said polynucleotide sequence to be transcribed from aninducible promoter, and in some instances, the gene product is selectedfrom the group consisting of: (a) an immunoglobulin heavy chain; (b) animmunoglobulin light chain; (c) manganese peroxidase; and (d) a fragmentof any of (a)-(c); or is an infliximab heavy or light chain or afragment thereof, or has at least 80% or 90% amino acid sequenceidentity with SEQ ID NO:30 or SEQ ID NO:31 across at least 50% or 80% ofthe length of SEQ ID NO:30 or SEQ ID NO:31, respectively, or has theamino acid sequence of SEQ ID NO:30 or SEQ ID NO:31; or has at least 80%or 90% amino acid sequence identity with SEQ ID NO:13, SEQ ID NO:15, orSEQ ID NO:23 across at least 50% or 80% of the length of SEQ ID NO:13,SEQ ID NO:15, or SEQ ID NO:23, respectively, or has the amino acidsequence of SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:23; or is apolypeptide selected from the group consisting of arabinose- andxylose-utilization enzymes such as xylose isomerase; or is a polypeptideselected from the group consisting of lignin-degrading peroxidases, suchas manganese peroxidase or versatile peroxidase.

Methods of producing products are also provided by the invention, suchas by growing a culture of a host cell of the invention as describedabove; and adding an inducer of at least one inducible promoter to theculture; a gene product or a multimeric product produced by this methodis also provided by the invention, and in some embodiments is anantibody, or in more particular instances, is an aglycosylated antibody,a chimeric antibody, or a human antibody.

Also provided by the systems and methods of the invention are kitscomprising a host cell, the host cell comprising two or more types ofexpression constructs, wherein the expression construct of each typecomprises an inducible promoter; and kits comprising a gene product or amultimeric product produced by growing a host cell of the invention andadding at least one inducer to the culture, where in some embodimentsthe multimeric product is an antibody, or in more particular instances,is an aglycosylated antibody, a chimeric antibody, or a human antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of the inducible coexpression system,which includes a host cell (1) comprising two different inducibleexpression vectors (3) and (4), which express different gene productsupon application of inducers (5), forming a multimeric product (6).

FIG. 2 is a schematic illustration of a particular use of the induciblecoexpression system, in which the E. coli host cell genome (2) encodes acytoplasmic form of the disulfide isomerase DsbC which lacks a signalpeptide; the expression vector pBAD24 (3) provides L-arabinose-inducibleexpression of an immunoglobulin heavy chain, and the expression vectorpPRO33 (4) provides propionate-inducible expression of an immunoglobulinlight chain; forming upon induction (5) the multimeric antibody product(6).

FIG. 3 shows the result of coexpression of immunoglobulin heavy andlight chains in bacterial cells. SHuffle® Express and BL21 cellscontaining both the pBAD24-HC and pPRO33-LC inducible expression vectorswere induced by growth in L-arabinose and propionate. Soluble proteinextracts from induced cells and uninduced controls were separated by SDSgel electrophoresis under reducing conditions on a 4-12% Bis-Tris gel.Lane 1: Induced SHuffle® Express. Lane 2: Uninduced SHuffle® Express.Lane 3: Induced BL21. Lane 4: Uninduced BL21. Arrows indicate a proteinband (IgG1 heavy chain) at 51 kDa and another protein band (IgG1 lightchain) at 26 kDa; these bands are present in the induced cells but notin the uninduced cells.

FIG. 4 shows the result of coexpression of immunoglobulin heavy andlight chains in bacterial cells. The same soluble protein extracts frominduced and uninduced SHuffle® Express and BL21 cells containing boththe pBAD24-HC and pPRO33-LC inducible expression vectors, as describedfor FIG. 3, were separated by gel electrophoresis under native(non-reducing) conditions on a 10-20% Tris-Glycine gel. Lane 1: InducedSHuffle® Express. Lane 2: Uninduced SHuffle® Express. Lane 3: InducedBL21. Lane 4: Uninduced BL21. Arrow indicates a protein band (IgG1antibody comprising heavy and light chains) at 154 kDa; this band ispresent in the induced SHuffle® Express cells, but is significantlyreduced or absent in the induced BL21 cells and in the uninduced cells.

FIG. 5 shows the result of coexpression, in bacterial cells, ofmanganese peroxidase (MnP) and protein disulfide isomerase (PDI) in thepresence of heme. SHuffle® Express cells, containing both thepPRO33-MnP-ChuA and pBAD24-PDI inducible expression vectors, wereinduced by growth in L-arabinose and propionate. Soluble proteinextracts from uninduced and induced cells were separated by gelelectrophoresis under reducing conditions on a 10% Bis-Glycine gel.

Markers: Bio-Rad Precision Plus Protein ™ Standard (pre-stained) Lane 1:Not Induced (no hemin, no propionate, no arabinose) Lane 2: 50 mMpropionate 0.002% arabinose Lane 3: 25 mM propionate 0.002% arabinoseLane 4: 12.5 mM propionate 0.002% arabinose Lane 5: 50 mM propionate0.01% arabinose Lane 6: 25 mM propionate 0.01% arabinose Lane 7: 12.5 mMpropionate 0.01% arabinose Lane 8: 50 mM propionate 0.05% arabinose Lane9: 25 mM propionate 0.05% arabinose Lane 10: 12.5 mM propionate 0.05%arabinoseThe arrows indicate protein bands, MnP at 39 kDa and PDI at 53 kDa;these bands are present in the SHuffle® Express cells most stronglyunder certain of the inducing conditions, but are significantly reducedin the uninduced cells.

FIG. 6 shows the result of coexpression, in bacterial cells, of analternate mature form of manganese peroxidase (MnP_FT) and proteindisulfide isomerase (PDI) in the presence of heme. SHuffle® Expresscells, containing both the pBAD24-MnP_FT-ChuA and pPRO33-PDI inducibleexpression vectors, were induced by growth in 0.1% L-arabinose and 50 mMpropionate. Soluble protein extracts from uninduced and induced cellswere separated by gel electrophoresis under reducing conditions on a 10%Bis-Glycine gel.

-   -   Lane 1: Molecular Weight Markers        -   Bio-Rad Precision Plus Protein™ Standard (pre-stained)    -   Lane 2: Induced Coexpression (0.1% L-arabinose, 50 mM        propionate)    -   Lane 3: Not Induced (no hemin, no L-arabinose, no propionate)    -   Lane 4: Control Induced (no protein-coding inserts)    -   Lane 5: Control Not Induced (no protein-coding inserts)

DETAILED DESCRIPTION OF THE INVENTION

The problem of incompatible coexpression system components is addressedby development of coordinated bacterial coexpression systems whichutilize compatible homogenously inducible promoter systems located onseparate expression constructs and, in some embodiments, activated bydifferent inducers. The advantages of the present invention include,without limitation: 1) improved compatibility of components within theinducible coexpression system; 2) inducible expression of gene productsthat together form multimers, or other combinations of gene products(coexpression of two or more gene products); 3) improved control of geneproduct coexpression by independently titratible induction; 4) improvedexpression of gene product complexes and other products that aredifficult to express such as multimeric products and products formingdisulfide bonds; 5) streamlined optimization of gene productcoexpression.

Coexpressed Gene Products.

The inducible coexpression systems of the invention are designed tocoexpress two or more different gene products that contribute to adesired product. The desired product can be a multimer, formed fromcoexpressed gene products, or coexpression can be used to produce acombination of the desired product plus an additional product orproducts that assist in expression of the desired product.

A ‘multimeric product’ refers to a set of gene products that coassembleto carry out the function of the multimeric product, and does not referto transitory associations between gene products and other molecules,such as modifying enzymes (kinases, peptidases, and the like),chaperones, transporters, etc. In certain embodiments of the invention,the multimeric products are heteromultimers. In many embodiments, thecoexpressed gene products will be polypeptides that are subunits ofmultimeric proteins. However, it is also possible to use the induciblecoexpression systems of the invention to coexpress multiple differentnon-coding RNA molecules, or a combination of polypeptide and non-codingRNA gene products. Non-coding RNA molecules, also callednon-protein-coding RNA (npcRNA), non-messenger RNA (nmRNA), andfunctional RNA (fRNA), include many different types of RNA moleculessuch as microRNAs that are not messenger RNAs and thus are not templatesfor the formation of polypeptides through translation.

Many biologically important products are formed from combinations ofdifferent polypeptide chains. In addition to antibodies and antibodyfragments, other multimeric products that can be produced by theinducible coexpression methods of the invention include G-coupledprotein receptors and ligand-gated ion channels such as nicotinicacetylcholine receptors, GABA_(A) receptors, glycine receptors,serotonin receptors, glutamate receptors, and ATP-gated receptors suchas P2X receptors. The botulinum neurotoxin (often referred to as BoTN,BTX, or as one of its commercially available forms, BOTOX®(onabotulinumtoxinA)) is formed from a heavy chain and a light chain,linked by a disulfide bond (Simpson et al., “The role of the interchaindisulfide bond in governing the pharmacological actions of botulinumtoxin”, J Pharmacol Exp Ther 2004 March; 308(3): 857-864, Epub 2003 Nov.14). Another example of a product formed from different polypeptidechains is insulin, which in eukaryotes is first translated as a singlepolypeptide chain, folded, and then cleaved ultimately into twopolypeptide chains held together by disulfide bonds. Efficientproduction of botulinum neurotoxin or of mature insulin in a single hostcell are examples of uses of the inducible coexpression methods of theinvention.

The methods of the invention are designed to produce gene products thathave been correctly folded and/or assembled into functional products,and that have a desired number of disulfide bonds in the desiredlocations within such functional products (which can be determined bymethods such as that of Example 11). The number of disulfide bonds for agene product such as a polypeptide is the total number of intramolecularand intermolecular bonds formed by that gene product when it is presentin a desired functional product. For example, a light chain of a humanIgG antibody typically has three disufide bonds (two intramolecularbonds and one intermolecular bond), and a heavy chain of a human IgGantibody typically has seven disufide bonds (four intramolecular bondsand three intermolecular bonds). In some embodiments, desired geneproducts are coexpressed with other gene products, such as chaperones,that are beneficial to the production of the desired gene product.Chaperones are proteins that assist the non-covalent folding orunfolding, and/or the assembly or disassembly, of other gene products,but do not occur in the resulting monomeric or multimeric gene productstructures when the structures are performing their normal biologicalfunctions (having completed the processes of folding and/or assembly).Chaperones can be expressed from an inducible promoter or a constitutivepromoter within an expression construct, or can be expressed from thehost cell chromosome; preferably, expression of chaperone protein(s) inthe host cell is at a sufficiently high level to produce coexpressedgene products that are properly folded and/or assembled into the desiredproduct. Examples of chaperones present in E. coli host cells are thefolding factors DnaK/DnaJ/GrpE, DsbC/DsbG, GroEL/GroES, IbpA/IbpB, Skp,Tig (trigger factor), and FkpA, which have been used to prevent proteinaggregation of cytoplasmic or periplasmic proteins. DnaK/DnaJ/GrpE,GroEL/GroES, and ClpB can function synergistically in assisting proteinfolding and therefore expression of these chaperones in combinations hasbeen shown to be beneficial for protein expression (Makino et al.,“Strain engineering for improved expression of recombinant proteins inbacteria”, Microb Cell Fact 2011 May 14; 10: 32). When expressingeukaryotic proteins in prokaryotic host cells, a eukaryotic chaperoneprotein, such as protein disulfide isomerase (PDI) from the same or arelated eukaryotic species, is coexpressed or inducibly coexpressed withthe desired gene product in certain embodiments of the invention.

Inducible Promoters.

The following is a description of inducible promoters that can be usedin expression constructs for coexpression of gene products, along withsome of the genetic modifications that can be made to host cells thatcontain such expression constructs. Examples of these induciblepromoters and related genes are, unless otherwise specified, fromEscherichia coli (E. coli) strain MG1655 (American Type CultureCollection deposit ATCC 700926), which is a substrain of E. coli K-12(American Type Culture Collection deposit ATCC 10798). Table 1 lists thegenomic locations, in E. coli MG1655, of the nucleotide sequences forthese examples of inducible promoters and related genes. Nucleotide andother genetic sequences, referenced by genomic location as in Table 1,are expressly incorporated by reference herein. Additional informationabout E. coli promoters, genes, and strains described herein can befound in many public sources, including the online EcoliWiki resource,located at ecoliwiki.net.

Arabinose Promoter.

(As used herein, ‘arabinose’ means L-arabinose.) Several E. coli operonsinvolved in arabinose utilization are inducible by arabinose—araBAD,araC, araE, and araFGH—but the terms ‘arabinose promoter’ and ‘arapromoter’ are typically used to designate the araBAD promoter. Severaladditional terms have been used to indicate the E. coli araBAD promoter,such as P_(ara), P_(araB), P_(araBAD), and P_(BAD). The use herein of‘ara promoter’ or any of the alternative terms given above, means the E.coli araBAD promoter. As can be seen from the use of another term,‘araC-araBAD promoter’, the araBAD promoter is considered to be part ofa bidirectional promoter, with the araBAD promoter controllingexpression of the araBAD operon in one direction, and the araC promoter,in close proximity to and on the opposite strand from the araBADpromoter, controlling expression of the araC coding sequence in theother direction. The AraC protein is both a positive and a negativetranscriptional regulator of the araBAD promoter. In the absence ofarabinose, the AraC protein represses transcription from P_(BAD), but inthe presence of arabinose, the AraC protein, which alters itsconformation upon binding arabinose, becomes a positive regulatoryelement that allows transcription from P_(BAD). The araBAD operonencodes proteins that metabolize L-arabinose by converting it, throughthe intermediates L-ribulose and L-ribulose-phosphate, toD-xylulose-5-phosphate. For the purpose of maximizing induction ofexpression from an arabinose-inducible promoter, it is useful toeliminate or reduce the function of AraA, which catalyzes the conversionof L-arabinose to L-ribulose, and optionally to eliminate or reduce thefunction of at least one of AraB and AraD, as well. Eliminating orreducing the ability of host cells to decrease the effectiveconcentration of arabinose in the cell, by eliminating or reducing thecell's ability to convert arabinose to other sugars, allows morearabinose to be available for induction of the arabinose-induciblepromoter. The genes encoding the transporters which move arabinose intothe host cell are araE, which encodes the low-affinity L-arabinoseproton symporter, and the araFGH operon, which encodes the subunits ofan ABC superfamily high-affinity L-arabinose transporter. Other proteinswhich can transport L-arabinose into the cell are certain mutants of theLacY lactose permease: the LacY(A177C) and the LacY(A177V) proteins,having a cysteine or a valine amino acid instead of alanine at position177, respectively (Morgan-Kiss et al., “Long-term and homogeneousregulation of the Escherichia coli araBAD promoter by use of a lactosetransporter of relaxed specificity”, Proc Natl Acad Sci USA 2002 May 28;99(11): 7373-7377). In order to achieve homogenous induction of anarabinose-inducible promoter, it is useful to make transport ofarabinose into the cell independent of regulation by arabinose. This canbe accomplished by eliminating or reducing the activity of the AraFGHtransporter proteins and altering the expression of araE so that it isonly transcribed from a constitutive promoter. Constitutive expressionof araE can be accomplished by eliminating or reducing the function ofthe native araE gene, and introducing into the cell an expressionconstruct which includes a coding sequence for the AraE proteinexpressed from a constitutive promoter. Alternatively, in a cell lackingAraFGH function, the promoter controlling expression of the host cell'schromosomal araE gene can be changed from an arabinose-induciblepromoter to a constitutive promoter. In similar manner, as additionalalternatives for homogenous induction of an arabinose-induciblepromoter, a host cell that lacks AraE function can have any functionalAraFGH coding sequence present in the cell expressed from a constitutivepromoter. As another alternative, it is possible to express both thearaE gene and the araFGH operon from constitutive promoters, byreplacing the native araE and araFGH promoters with constitutivepromoters in the host chromosome. It is also possible to eliminate orreduce the activity of both the AraE and the AraFGH arabinosetransporters, and in that situation to use a mutation in the LacYlactose permease that allows this protein to transport arabinose. Sinceexpression of the lacY gene is not normally regulated by arabinose, useof a LacY mutant such as LacY(A177C) or LacY(A177V), will not lead tothe ‘all or none’ induction phenomenon when the arabinose-induciblepromoter is induced by the presence of arabinose. Because theLacY(A177C) protein appears to be more effective in transportingarabinose into the cell, use of polynucleotides encoding the LacY(A177C)protein is preferred to the use of polynucleotides encoding theLacY(A177V) protein.

Propionate Promoter.

The ‘propionate promoter’ or ‘prp promoter’ is the promoter for the E.coli prpBCDE operon, and is also called P_(prpB). Like the am promoter,the prp promoter is part of a bidirectional promoter, controllingexpression of the prpBCDE operon in one direction, and with the prpRpromoter controlling expression of the prpR coding sequence in the otherdirection. The PrpR protein is the transcriptional regulator of the prppromoter, and activates transcription from the prp promoter when thePrpR protein binds 2-methylcitrate (‘2-MC’). Propionate (also calledpropanoate) is the ion, CH₃CH₂COO , of propionic acid (or ‘propanoicacid’), and is the smallest of the ‘fatty’ acids having the generalformula H(CH₂)_(n)COOH that shares certain properties of this class ofmolecules: producing an oily layer when salted out of water and having asoapy potassium salt. Commercially available propionate is generallysold as a monovalent cation salt of propionic acid, such as sodiumpropionate (CH₃CH₂COONa), or as a divalent cation salt, such as calciumpropionate (Ca(CH₃CH₂C00)₂). Propionate is membrane-permeable and ismetabolized to 2-MC by conversion of propionate to propionyl-CoA by PrpE(propionyl-CoA synthetase), and then conversion of propionyl-CoA to 2-MCby PrpC (2-methylcitrate synthase). The other proteins encoded by theprpBCDE operon, PrpD (2-methylcitrate dehydratase) and PrpB(2-methylisocitrate lyase), are involved in further catabolism of 2-MCinto smaller products such as pyruvate and succinate. In order tomaximize induction of a propionate-inducible promoter by propionateadded to the cell growth medium, it is therefore desirable to have ahost cell with PrpC and PrpE activity, to convert propionate into 2-MC,but also having eliminated or reduced PrpD activity, and optionallyeliminated or reduced PrpB activity as well, to prevent 2-MC from beingmetabolized. Another operon encoding proteins involved in 2-MCbiosynthesis is the scpA-argK-scpBC operon, also called the sbm-ygfDGHoperon. These genes encode proteins required for the conversion ofsuccinate to propionyl-CoA, which can then be converted to 2-MC by PrpC.Elimination or reduction of the function of these proteins would removea parallel pathway for the production of the 2-MC inducer, and thusmight reduce background levels of expression of a propionate-induciblepromoter, and increase sensitivity of the propionate-inducible promoterto exogenously supplied propionate. It has been found that a deletion ofsbm-ygfD-ygfG-ygfH-ygfI, introduced into E. coli BL21(DE3) to createstrain JSB (Lee and Keasling, “A propionate-inducible expression systemfor enteric bacteria”, Appl Environ Microbiol 2005 November; 71(11):6856-6862), was helpful in reducing background expression in the absenceof exogenously supplied inducer, but this deletion also reduced overallexpression from the prp promoter in strain JSB. It should be noted,however, that the deletion sbm-ygfD-ygfG-ygfH-ygfI also apparentlyaffects ygfI, which encodes a putative LysR-family transcriptionalregulator of unknown function. The genes sbm-ygfDGH are transcribed asone operon, and ygfI is transcribed from the opposite strand. The 3′ends of the ygfH and ygfI coding sequences overlap by a few base pairs,so a deletion that takes out all of the sbm-ygfDGH operon apparentlytakes out ygfI coding function as well. Eliminating or reducing thefunction of a subset of the sbm-ygfDGH gene products, such as YgfG (alsocalled ScpB, methylmalonyl-CoA decarboxylase), or deleting the majorityof the sbm-ygfDGH (or scpA-argK-scpBC) operon while leaving enough ofthe 3′ end of the ygfH (or scpC) gene so that the expression of ygfI isnot affected, could be sufficient to reduce background expression from apropionate-inducible promoter without reducing the maximal level ofinduced expression.

Rhamnose Promoter.

(As used herein, ‘rhamnose’ means L-rhamnose.) The ‘rhamnose promoter’or ‘rha promoter’, or P_(rhaSR), is the promoter for the E. coli rhaSRoperon. Like the ara and prp promoters, the rha promoter is part of abidirectional promoter, controlling expression of the rhaSR operon inone direction, and with the rhaBAD promoter controlling expression ofthe rhaBAD operon in the other direction. The rha promoter, however, hastwo transcriptional regulators involved in modulating expression: RhaRand RhaS. The RhaR protein activates expression of the rhaSR operon inthe presence of rhamnose, while RhaS protein activates expression of theL-rhamnose catabolic and transport operons, rhaBAD and rhaT,respectively (Wickstrum et al., “The AraC/XylS family activator RhaSnegatively autoregulates rhaSR expression by preventing cyclic AMPreceptor protein activation”, J Bacteriol 2010 January; 192(1):225-232). Although the RhaS protein can also activate expression of therhaSR operon, in effect RhaS negatively autoregulates this expression byinterfering with the ability of the cyclic AMP receptor protein (CRP) tocoactivate expression with RhaR to a much greater level. The rhaBADoperon encodes the rhamnose catabolic proteins RhaA (L-rhamnoseisomerase), which converts L-rhamnose to L-rhamnulose; RhaB(rhamnulokinase), which phosphorylates L-rhamnulose to formL-rhamnulose-1-P; and RhaD (rhamnulose-1-phosphate aldolase), whichconverts L-rhamnulose-1-P to L-lactaldehyde and DHAP (dihydroxyacetonephosphate). To maximize the amount of rhamnose in the cell available forinduction of expression from a rhamnose-inducible promoter, it isdesirable to reduce the amount of rhamnose that is broken down bycatalysis, by eliminating or reducing the function of RhaA, oroptionally of RhaA and at least one of RhaB and RhaD. E. coli cells canalso synthesize L-rhamnose from alpha-D-glucose-1-P through theactivities of the proteins RmlA, RmlB, RmlC, and RmlD (also called RfbA,RfbB, RfbC, and RfbD, respectively) encoded by the rmlBDACX (or rfbBDACXoperon. To reduce background expression from a rhamnose-induciblepromoter, and to enhance the sensitivity of induction of therhamnose-inducible promoter by exogenously supplied rhamnose, it couldbe useful to eliminate or reduce the function of one or more of theRmlA, RmlB, RmlC, and RmlD proteins. L-rhamnose is transported into thecell by RhaT, the rhamnose permease or L-rhamnose:proton symporter. Asnoted above, the expression of RhaT is activated by the transcriptionalregulator RhaS. To make expression of RhaT independent of induction byrhamnose (which induces expression of RhaS), the host cell can bealtered so that all functional RhaT coding sequences in the cell areexpressed from constitutive promoters. Additionally, the codingsequences for RhaS can be deleted or inactivated, so that no functionalRhaS is produced. By eliminating or reducing the function of RhaS in thecell, the level of expression from the rhaSR promoter is increased dueto the absence of negative autoregulation by RhaS, and the level ofexpression of the rhamnose catalytic operon rhaBAD is decreased, furtherincreasing the ability of rhamnose to induce expression from the rhapromoter.

Xylose promoter. (As used herein, ‘xylose’ means D-xylose.) The xylosepromoter, or ‘xyl promoter’, or P_(xylA), means the promoter for the E.coli xylAB operon. The xylose promoter region is similar in organizationto other inducible promoters in that the xylAB operon and the xylFGHRoperon are both expressed from adjacent xylose-inducible promoters inopposite directions on the E. coli chromosome (Song and Park,“Organization and regulation of the D-xylose operons in Escherichia coliK-12: XylR acts as a transcriptional activator”, J Bacteriol. 1997November; 179(22): 7025-7032). The transcriptional regulator of both theP_(xylA) and P_(xylF) promoters is XylR, which activates expression ofthese promoters in the presence of xylose. The xylR gene is expressedeither as part of the xylFGHR operon or from its own weak promoter,which is not inducible by xylose, located between the xylH and xylRprotein-coding sequences. D-xylose is catabolized by XylA (D-xyloseisomerase), which converts D-xylose to D-xylulose, which is thenphosphorylated by XylB (xylulokinase) to form D-xylulose-5-P. Tomaximize the amount of xylose in the cell available for induction ofexpression from a xylose-inducible promoter, it is desirable to reducethe amount of xylose that is broken down by catalysis, by eliminating orreducing the function of at least XylA, or optionally of both XylA andXylB. The xylFGHR operon encodes XylF, XylG, and XylH, the subunits ofan ABC superfamily high-affinity D-xylose transporter. The xylE gene,which encodes the E. coli low-affinity xylose-proton symporter,represents a separate operon, the expression of which is also inducibleby xylose. To make expression of a xylose transporter independent ofinduction by xylose, the host cell can be altered so that all functionalxylose transporters are expressed from constitutive promoters. Forexample, the xylFGHR operon could be altered so that the xylFGH codingsequences are deleted, leaving XylR as the only active protein expressedfrom the xylose-inducible P_(xylF) promoter, and with the xylE codingsequence expressed from a constitutive promoter rather than its nativepromoter. As another example, the xylR coding sequence is expressed fromthe P_(xylA) or the P_(xylF) promoter in an expression construct, whileeither the xylFGHR operon is deleted and xylE is constitutivelyexpressed, or alternatively an xylFGH operon (lacking the xylR codingsequence since that is present in an expression construct) is expressedfrom a constitutive promoter and the xylE coding sequence is deleted oraltered so that it does not produce an active protein.

Lactose promoter. The term ‘lactose promoter’ refers to thelactose-inducible promoter for the lacZYA operon, a promoter which isalso called lacZpl; this lactose promoter is located at ca.365603-365568 (minus strand, with the RNA polymerase binding (‘-35’)site at ca. 365603-365598, the Pribnow box (‘-10’) at 365579-365573, anda transcription initiation site at 365567) in the genomic sequence ofthe E. coli K-12 substrain MG1655 (NCBI Reference Sequence NC 000913.2,11 Jan. 2012). In some embodiments, inducible coexpression systems ofthe invention can comprise a lactose-inducible promoter such as thelacZYA promoter. In other embodiments, the inducible coexpressionsystems of the invention comprise one or more inducible promoters thatare not lactose-inducible promoters.

TABLE 1 Genomic Locations of E. coli Inducible Promoters and RelatedGenes[1] Genomic Promoter or Gene Location: Comments: araBAD promoter[2] (ca. 70165)- Smith and Schleif[3]: RNA pol[4] binding (‘−35’) 70074(minus 70110-70104, Pribnow box (‘−10’) 70092-70085 strand) araBADoperon 70075-65855 Smith and Schleif[3]: transcript start 70075, araB(minus strand) ATG 70048; NCBI: araB end of TAA 68348; araA ATG 68337,end of TAA 66835; araD ATG 66550, end of TAA 65855 araC promoter [2](ca. 70166)- Smith and Schleif[3]: RNA pol binding (‘−35’) 70210- 70241(plus 7021, Pribnow box (‘−10’) 70230-70236 strand) araC gene70242-71265 Miyada[5]: transcript start 70242, araC ATG 70387; (plusstrand) NCBI: end of TAA 71265 araE promoter [2] (ca. 2980349)- Stonerand Schleif[6]: CRP binding 2980349- 2980231 2980312, RNA pol binding(‘−35’) 2980269-2980264, (minus strand) Pribnow box (‘−10’)2980244-2980239 araE gene 2980230- Stoner and Schleif[6]: transcriptstart 2980230, ATG 2978786 (minus 2980204; NCBI: end of TGA 2978786strand) araFGH promoter [2] (ca. 1984423)- Hendrickson[7]: AraC bindingca. 1984423-ca. 1984264 1984414 and 1984326-1984317, CRP binding (minusstrand) 1984315-1984297, RNA pol binding (‘−35’) 1984294- 1984289,Pribnow box (‘−10’) 1984275-1984270 araFGH operon 1984263-Hendrickson[7]: transcript start 1984263; NCBI: 1980578 (minus araF ATG1984152, end of TAA 1983163; araG ATG strand) 1983093, end of TGA1981579; araH ATG 1981564, end of TGA 1980578 lacY gene 362403-361150Expressed as part of the lacZYA operon. NCBI: ATG (minus strand) 362403,end of TAA 361150 prpBCDE [2] ca. 347790- Keasling[8]: RNA pol binding(‘−24’) 347844-347848, promoter ca. 347870 (plus Pribnow box (‘−12’)347855-347859 strand) prpBCDE operon (ca. 347871)- Keasling[8]: inferredtranscript start ca. 347871, prpB 353816 (plus ATG 347906; NCBI: prpBend of TAA 348796; prpC strand) ATG 349236, end of TAA 350405; prpD ATG350439, end of TAA 351890; prpE ATG 351930, end of TAG 353816 prpRpromoter [2] ca. 347789- Keasling[8]: CRP binding 347775-347753, RNA polca. 347693 binding (‘−35’) 347728-347723, Pribnow box (‘−10’) (minusstrand) 347707-347702 prpR gene (ca. 347692)- Keasling[8]: inferredtranscript start ca. 347692, prpR 346081 (minus ATG 347667; NCBI: end ofTGA 346081 strand) scpA-argK-scpBC 3058872- NCBI: scpA ATG 3058872, endof TAA 3061016; (or sbm-ygfDGH) 3064302 (plus argK ATG 3061009, end ofTAA 3062004; scpB ATG operon strand) 3062015, end of TAA 3062800; scpCATG 3062824, end of TAA 3064302 rhaBAD promoter [2] (ca. 4095605)-Wickstrum[9]: CRP binding 4095595-4095580, RNA 4095496 pol binding(‘−35’) 4095530-4095525, Pribnow box (minus strand) (‘−10’)4095506-4095501 rhaBAD operon 4095495- Wickstrum[9]: transcript start4095495, rhaB ATG 4091471 (minus 4095471; NCBI: rhaB end of TGA 4094002;rhaA strand) ATG 4094005, end of TAA 4092746; rhaD ATG 4092295, end ofTAA 4091471 rhaSR promoter [2] (ca. 4095606)- Wickstrum[9]: CRP binding4095615-4095630, RNA 4095733 (plus pol binding (‘−35’) 4095699-4095704,Pribnow box strand) (‘−10’) 4095722-4095727 rhaSR operon 4095734-Wickstrum[9]: transcript start 4095734, rhaS ATG 4097517 (plus 4095759;NCBI: rhaS end of TAA 4096595; rhaR strand) ATG 4096669, end of TAA4097517 rfbBDACX (or 2111085- NCBI: rfbB GTG 2111085, end of TAA2110000; rfbD rmlBDACX) 2106361 (minus ATG 2110000, end of TAA 2109101;rfbA ATG operon strand) 2109043, end of TAA 2108162; rfbC ATG 2108162,end of TGA 2107605; rfbX ATG 2107608, end of TGA 2106361 rhaT promoter[2] (ca. 4098690)- Via[10]: CRP binding 4098690-4098675, RNA pol 4098590binding (‘−35’) 4098621-4098616, Pribnow box (‘−10’) (minus strand)4098601-4098596 rhaT gene 4098589- Via[10]: transcript start 4098589,rhaT ATG 4098548; 4097514 (minus NCBI: rhaT end of TAA 4097514 strand)xylAB promoter [2] (ca. 3728960)- Song and Park[11]: CRP binding3728919-3728901, 3728831 RNA pol binding (‘−35’) 3728865-3728860,Pribnow (minus strand) box (‘−10’) 3728841-3728836 xylAB operon 3728830-Song and Park[11]: transcript start 3728830, xylA 3725940 (minus ATG3728788; NCBI: xylA end of TAA 3727466; strand) xylB ATG 3727394, end ofTAA 3725940 xylFGHR [2](ca. 3728961)- Song and Park[11]: RNA pol binding(‘−35’) 3729058- promoter 3729091 (plus 3729063, Pribnow box (‘−10’)3729080-3729085 strand) xylFGHR operon 3729092- Song and Park[11]:transcript start 3729092, xylF 3734180 (plus ATG 3729154; NCBI: xylF endof TAA 3730146, strand) xylG ATG 3730224, end of TGA 3731765; xylH ATG3731743, end of TGA 3732924; xylR ATG 3733002, end of TAG 3734180 xylEpromoter [2] ca. 4240482- Davis and Henderson[12]: possible Pribnow boxca. 4240320 (‘−10’) 4240354-4240349, possible Pribnow box (‘−10’) (minusstrand) 4240334-4240329 xylE gene (ca. 4240319)- Davis andHenderson[12]: inferred transcript start ca. 4238802 (minus 4240319,xylE ATG 4240277, end of TAA 4238802 strand) Notes for Table 1: [1] Allgenomic sequence locations refer to the genomic sequence of E. coli K-12substrain MG1655, provided by the National Center for BiotechnologyInformation (NCBI) as NCBI Reference Sequence NC_000913.2, 11-JAN-2012.[2] The location of the 5′ (or ‘upstream’) end of the promoter region isapproximated; for ‘bidirectional’ promoters, a nucleotide sequencelocation that is approximately equidistant between the transcriptionstart sites is selected as the designated 5′ ‘end’ for both of theindividual promoters. In practice, the promoter portion of an expressionconstruct can have somewhat less sequence at its 5′ end than thepromoter sequences as indicated in the table, or it can have anucleotide sequence that includes additional sequence from the region 5′(or ‘upstream’) of the promoter sequences as indicated in the table, aslong as it retains the ability to promote transcription of a downstreamcoding sequence in an inducible fashion. [3] Smith and Schleif,“Nucleotide sequence of the L-arabinose regulatory region of Escherichiacoli K12”, J Biol Chem 1978 Oct 10; 253(19): 6931-6933. [4] ‘RNA pol’indicates RNA polymerase throughout the table. [5] Miyada, et al., “DNAsequence of the araC regulatory gene from Escherichia coli B/r”, NucleicAcids Res 1980 Nov 25; 8(22): 5267-5274. [6] Stoner and Schleif, “E.coli araE regulatory region araE codes for the low affinity L-arabinoseuptake protein”, GenBank Database Accession X00272.1, revision date06-JUL-1989. [7] Hendrickson et al., “Sequence elements in theEscherichia coli araFGH promoter”, J Bacteriol 1992 Nov; 174(21):6862-6871. [8] U.S. Pat. No. 8,178,338 B2; May 15, 2012; Keasling, Jay;FIG. 9. [9] Wickstrum et at., “The AraC/XylS family activator RhaSnegatively autoregulates rhaSR expression by preventing cyclic AMPreceptor protein activation”, J Bacteriol 2010 Jan; 192(1): 225-232.[10] Via et at., “Transcriptional regulation of the Escherichia colirhaT gene”, Microbiology 1996 Jul; 142(Pt 7): 1833-1840. [11] Song andPark, “Organization and regulation of the D-xylose operons inEscherichia coli K-12: XylR acts as a transcriptional activator”, JBacteriol. 1997 Nov; 179(22): 7025-7032. [12] Davis and Henderson, “Thecloning and DNA sequence of the gene xylE for xylose-proton symport inEscherichia coli K12”, J Biol Chem 1987 Oct 15; 262(29): 13928-13932.

Expression Constructs.

Expression constructs are polynucleotides designed for the expression ofone or more gene products of interest, and thus are not naturallyoccurring molecules. Expression constructs can be integrated into a hostcell chromosome, or maintained within the host cell as polynucleotidemolecules replicating independently of the host cell chromosome, such asplasmids or artificial chromosomes. An example of an expressionconstruct is a polynucleotide resulting from the insertion of one ormore polynucleotide sequences into a host cell chromosome, where theinserted polynucleotide sequences alter the expression of chromosomalcoding sequences. An expression vector is a plasmid expression constructspecifically used for the expression of one or more gene products. Oneor more expression constructs can be integrated into a host cellchromosome or be maintained on an extrachromosomal polynucleotide suchas a plasmid or artificial chromosome. The following are descriptions ofparticular types of polynucleotide sequences that can be used inexpression constructs for the coexpression of gene products.

Origins of Replication.

Expression constructs must comprise an origin of replication, alsocalled a replicon, in order to be maintained within the host cell asindependently replicating polynucleotides. Different replicons that usethe same mechanism for replication cannot be maintained together in asingle host cell through repeated cell divisions. As a result, plasmidscan be categorized into incompatibility groups depending on the originof replication that they contain, as shown in Table 2.

TABLE 2 Origins of Replication and Representative Plasmids for Use inExpression Constructs [1] Incompatibility Origin of Copy RepresentativePlasmids Group: Replication: Number: (ATCC Deposit No.): colE1, pMB1colE1 15-20 colE1 (ATCC 27138) pMB1 15-20 pBR322 (ATCC 31344) Modified500-700 pUC9 (ATCC 37252) pMB1 IncFII, pT181 R1(ts) 15-120 pMOB45 (ATCC37106) F, P1, p15A, p15A 18-22 pACYC177 (ATCC 37031); pSC101, R6K,pACYC184 (ATCC 37033); RK2 [2] pPRO33 (Addgene 17810)[3] pSC101 ~5pSC101 (ATCC 37032): pGBM1 (ATCC 87497) RK2  4-7 [2] RK2 (ATCC 37125)CloDF13 [4] CloDF13 20-40 [4] pCDFDuet ™-1 (EMD Millipore Catalog No.71340-3) ColA [4] ColA 20-40 [4] pCOLADuet ™-1 (EMD Millipore CatalogNo. 71406-3) RSF1030[4] RSF1030  >100 [4] pRSFDuet ™-1 (EMD (alsoMillipore Catalog called No. 71341-3) NTP1) Notes for Table 2: [1]Adapted from www.bio.davidson.edu/courses/Molbio/Protocols/ORIs.html,and Sambrook and Russell, “Molecular Cloning: A laboratory manual”,3^(rd) Ed., Cold Spring Harbor Laboratory Press, 2001. [2] Kües andStahl, “Replication of plasmids in gram-negative bacteria”, MicrobiolRev 1989 Dec; 53(4): 491-516. [3] The pPRO33 plasmid (U.S. Pat. No.8,178,338 B2; May 15, 2012; Keasling, Jay) is available from Addgene(www.addgene.org) as Addgene plasmid 17810. [4]openwetware.org/wiki/CH391L/S12/Origins_of_Replication; accessed 3 Aug.2013.

Origins of replication can be selected for use in expression constructson the basis of incompatibility group, copy number, and/or host range,among other criteria. As described above, if two or more differentexpression constructs are to be used in the same host cell for thecoexpression of multiple gene products, it is best if the differentexpression constructs contain origins of replication from differentincompatibility groups: a pMB1 replicon in one expression construct anda p15A replicon in another, for example. The average number of copies ofan expression construct in the cell, relative to the number of hostchromosome molecules, is determined by the origin of replicationcontained in that expression construct. Copy number can range from a fewcopies per cell to several hundred (Table 2). In one embodiment of theinvention, different expression constructs are used which compriseinducible promoters that are activated by the same inducer, but whichhave different origins of replication. By selecting origins ofreplication that maintain each different expression construct at acertain approximate copy number in the cell, it is possible to adjustthe levels of overall production of a gene product expressed from oneexpression construct, relative to another gene product expressed from adifferent expression construct. As an example, to coexpress subunits Aand B of a multimeric protein, an expression construct is created whichcomprises the colE1 replicon, the am promoter, and a coding sequence forsubunit A expressed from the am promoter: ‘colE1-P_(ara)-A’. Anotherexpression construct is created comprising the p15A replicon, the ampromoter, and a coding sequence for subunit B: ‘p15A-P_(ara)-B’. Thesetwo expression constructs can be maintained together in the same hostcells, and expression of both subunits A and B is induced by theaddition of one inducer, arabinose, to the growth medium. If theexpression level of subunit A needed to be significantly increasedrelative to the expression level of subunit B, in order to bring thestoichiometric ratio of the expressed amounts of the two subunits closerto a desired ratio, for example, a new expression construct for subunitA could be created, having a modified pMB1 replicon as is found in theorigin of replication of the pUC9 plasmid (‘pUC9ori’):pUC9ori-P_(ara)-A. Expressing subunit A from a high-copy-numberexpression construct such as pUC9ori-P_(ara)-A should increase theamount of subunit A produced relative to expression of subunit B fromp15A-P_(ara)-B. In a similar fashion, use of an origin of replicationthat maintains expression constructs at a lower copy number, such aspSC101, could reduce the overall level of a gene product expressed fromthat construct. Selection of an origin of replication can also determinewhich host cells can maintain an expression construct comprising thatreplicon. For example, expression constructs comprising the colE1 originof replication have a relatively narrow range of available hosts,species within the Enterobacteriaceae family, while expressionconstructs comprising the RK2 replicon can be maintained in E. coli,Pseudomonas aeruginosa, Pseudomonas putida, Azotobacter vinelandii, andAlcaligenes eutrophus, and if an expression construct comprises the RK2replicon and some regulator genes from the RK2 plasmid, it can bemaintained in host cells as diverse as Sinorhizobium mehloti,Agrobacterium tumefaciens, Caulobacter crescentus, Acinetobactercalcoaceticus, and Rhodobacter sphaeroides (Kües and Stahl, “Replicationof plasmids in grain-negative bacteria”, Microbiol Rev 1989 December;53(4): 491-516).

Similar considerations can be employed to create expression constructsfor inducible coexpression in eukaryotic cells. For example, the2-micron circle plasmid of Saccharomyces cerevisiae is compatible withplasmids from other yeast strains, such as pSR1 (ATCC Deposit Nos. 48233and 66069; Araki et al., “Molecular and functional organization of yeastplasmid pSR1”, J Mol Biol 1985 Mar. 20; 182(2): 191-203) and pKD1 (ATCCDeposit No. 37519; Chen et al., “Sequence organization of the circularplasmid pKD1 from the yeast Kluyveromyces drosophilarum”, Nucleic AcidsRes 1986 Jun. 11; 14(11): 4471-4481).

Selectable Markers.

Expression constructs usually comprise a selection gene, also termed aselectable marker, which encodes a protein necessary for the survival orgrowth of host cells in a selective culture medium. Host cells notcontaining the expression construct comprising the selection gene willnot survive in the culture medium. Typical selection genes encodeproteins that confer resistance to antibiotics or other toxins, or thatcomplement auxotrophic deficiencies of the host cell. One example of aselection scheme utilizes a drug such as an antibiotic to arrest growthof a host cell. Those cells that contain an expression constructcomprising the selectable marker produce a protein conferring drugresistance and survive the selection regimen. Some examples ofantibiotics that are commonly used for the selection of selectablemarkers (and abbreviations indicating genes that provide antibioticresistance phenotypes) are: ampicillin (Amp^(R)), chloramphenicol(Cml^(R) or Cm^(R)), kanamycin (Kan^(R)), spectinomycin (Spc^(R)),streptomycin (Str^(R)), and tetracycline (Tet^(R)). Many of therepresentative plasmids in Table 2 comprise selectable markers, such aspBR322 (Amp^(R), Tet^(R)); pMOB45 (Cm^(R), Tet^(R)); pACYC177 (Amp^(R),Kan^(R)); and pGBMl (Spc^(R), Str^(R)). The native promoter region for aselection gene is usually included, along with the coding sequence forits gene product, as part of a selectable marker portion of anexpression construct. Alternatively, the coding sequence for theselection gene can be expressed from a constitutive promoter.

Inducible Promoter.

As described herein, there are several different inducible promotersthat can be included in expression constructs as part of the induciblecoexpression systems of the invention. Preferred inducible promotersshare at least 80% polynucleotide sequence identity (more preferably, atleast 90% identity, and most preferably, at least 95% identity) to atleast 30 (more preferably, at least 40, and most preferably, at least50) contiguous bases of a promoter polynucleotide sequence as defined inTable 1 by reference to the E. coli K-12 substrain MG1655 genomicsequence, where percent polynucleotide sequence identity is determinedusing the methods of Example 13. Under ‘standard’ inducing conditions(see Example 5), preferred inducible promoters have at least 75% (morepreferably, at least 100%, and most preferably, at least 110%) of thestrength of the corresponding ‘wild-type’ inducible promoter of E. coliK-12 substrain MG1655, as determined using the quantitative PCR methodof De Mey et al. (Example 8). Within the expression construct, aninducible promoter is placed 5′ to (or ‘upstream of’) the codingsequence for the gene product that is to be inducibly expressed, so thatthe presence of the inducible promoter will direct transcription of thegene product coding sequence in a 5′ to 3′ direction relative to thecoding strand of the polynucleotide encoding the gene product.

Ribosome Binding Site.

For polypeptide gene products, the nucleotide sequence of the regionbetween the transcription initiation site and the initiation codon ofthe coding sequence of the gene product that is to be induciblyexpressed corresponds to the 5′ untranslated region (‘UTR’) of the mRNAfor the polypeptide gene product. Preferably, the region of theexpression construct that corresponds to the 5′ UTR comprises apolynucleotide sequence similar to the consensus ribosome binding site(RBS, also called the Shine-Dalgarno sequence) that is found in thespecies of the host cell. In prokaryotes (archaea and bacteria), the RBSconsensus sequence is GGAGG or GGAGGU, and in bacteria such as E. coli,the RBS consensus sequence is AGGAGG or AGGAGGU. The RBS is typicallyseparated from the initiation codon by 5 to 10 intervening nucleotides.In expression constructs, the RBS sequence is preferably at least 55%identical to the AGGAGGU consensus sequence, more preferably at least70% identical, and most preferably at least 85% identical, and isseparated from the initiation codon by 5 to 10 intervening nucleotides,more preferably by 6 to 9 intervening nucleotides, and most preferablyby 6 or 7 intervening nucleotides. The ability of a given RBS to producea desirable translation initiation rate can be calculated at the websitesalis.psu.edu/software/RBSLibraryCalculatorSearchMode, using the RBSCalculator; the same tool can be used to optimize a synthetic RBS for atranslation rate across a 100,000+ fold range (Salis, “The ribosomebinding site calculator”, Methods Enzymol 2011; 498: 19-42).

Multiple Cloning Site.

A multiple cloning site (MCS), also called a polylinker, is apolynucleotide that contains multiple restriction sites in closeproximity to or overlapping each other. The restriction sites in the MCStypically occur once within the MCS sequence, and preferably do notoccur within the rest of the plasmid or other polynucleotide construct,allowing restriction enzymes to cut the plasmid or other polynucleotideconstruct only within the MCS. Examples of MCS sequences are those inthe pBAD series of expression vectors, including pBAD18, pBAD18-Cm,pBAD18-Kan, pBAD24, pBAD28, pBAD30, and pBAD33 (Guzman et al., “Tightregulation, modulation, and high-level expression by vectors containingthe arabinose PBAD promoter”, J Bacteriol 1995 July; 177(14):4121-4130); or those in the pPRO series of expression vectors derivedfrom the pBAD vectors, such as pPRO18, pPRO18-Cm, pPRO18-Kan, pPRO24,pPRO30, and pPRO33 (U.S. Pat. No. 8,178,338 B2; May 15, 2012; Keasling,Jay). A multiple cloning site can be used in the creation of anexpression construct: by placing a multiple cloning site 3′ to (ordownstream of) a promoter sequence, the MCS can be used to insert thecoding sequence for a gene product to be coexpressed into the construct,in the proper location relative to the promoter so that transcription ofthe coding sequence will occur. Depending on which restriction enzymesare used to cut within the MCS, there may be some part of the MCSsequence remaining within the expression construct after the codingsequence or other polynucleotide sequence is inserted into theexpression construct. Any remaining MCS sequence can be upstream or, ordownstream of, or on both sides of the inserted sequence. A ribosomebinding site can be placed upstream of the MCS, preferably immediatelyadjacent to or separated from the MCS by only a few nucleotides, inwhich case the RBS would be upstream of any coding sequence insertedinto the MCS. Another alternative is to include a ribosome binding sitewithin the MCS, in which case the choice of restriction enzymes used tocut within the MCS will determine whether the RBS is retained, and inwhat relation to, the inserted sequences. A further alternative is toinclude a RBS within the polynucleotide sequence that is to be insertedinto the expression construct at the MCS, preferably in the properrelation to any coding sequences to stimulate initiation of translationfrom the transcribed messenger RNA.

Expression from Constitutive Promoters.

Expression constructs of the invention can also comprise codingsequences that are expressed from constitutive promoters. Unlikeinducible promoters, constitutive promoters initiate continual geneproduct production under most growth conditions. One example of aconstitutive promoter is that of the Tn3 bla gene, which encodesbeta-lactamase and is responsible for the ampicillin-resistance(Amp^(R)) phenotype conferred on the host cell by many plasmids,including pBR322 (ATCC 31344), pACYC177 (ATCC 37031), and pBAD24 (ATCC87399). Another constitutive promoter that can be used in expressionconstructs is the promoter for the E. coli lipoprotein gene, lpp, whichis located at positions 1755731-1755406 (plus strand) in E. coli K-12substrain MG1655 (Inouye and Inouye, “Up-promoter mutations in the lppgene of Escherichia coli”, Nucleic Acids Res 1985 May 10; 13(9):3101-3110). A further example of a constitutive promoter that has beenused for heterologous gene expression in E. coli is the trpLEDCBApromoter, located at positions 1321169-1321133 (minus strand) in E. coliK-12 substrain MG1655 (Windass et al., “The construction of a syntheticEscherichia coli trp promoter and its use in the expression of asynthetic interferon gene”, Nucleic Acids Res 1982 Nov. 11; 10(21):6639-6657). Constitutive promoters can be used in expression constructsfor the expression of selectable markers, as described herein, and alsofor the constitutive expression of other gene products useful for thecoexpression of the desired product. For example, transcriptionalregulators of the inducible promoters, such as AraC, PrpR, RhaR, andXylR, if not expressed from a bidirectional inducible promoter, canalternatively be expressed from a constitutive promoter, on either thesame expression construct as the inducible promoter they regulate, or adifferent expression construct. Similarly, gene products useful for theproduction or transport of the inducer, such as PrpEC, AraE, or Rha, orproteins that modify the reduction-oxidation environment of the cell, asa few examples, can be expressed from a constitutive promoter within anexpression construct. Gene products useful for the production ofcoexpressed gene products, and the resulting desired product, alsoinclude chaperone proteins, cofactor transporters, etc.

Signal Peptides.

Polypeptide gene products coexpressed by the methods of the inventioncan contain signal peptides or lack them, depending on whether it isdesirable for such gene products to be exported from the host cellcytoplasm into the periplasm, or to be retained in the cytoplasm,respectively. Signal peptides (also termed signal sequences, leadersequences, or leader peptides) are characterized structurally by astretch of hydrophobic amino acids, approximately five to twenty aminoacids long and often around ten to fifteen amino acids in length, thathas a tendency to form a single alpha-helix. This hydrophobic stretch isoften immediately preceded by a shorter stretch enriched in positivelycharged amino acids (particularly lysine). Signal peptides that are tobe cleaved from the mature polypeptide typically end in a stretch ofamino acids that is recognized and cleaved by signal peptidase. Signalpeptides can be characterized functionally by the ability to directtransport of a polypeptide, either co-translationally orpost-translationally, through the plasma membrane of prokaryotes (or theinner membrane of grain negative bacteria like E. coli), or into theendoplasmic reticulum of eukaryotic cells. The degree to which a signalpeptide enables a polypeptide to be transported into the periplasmicspace of a host cell like E. coli, for example, can be determined byseparating periplasmic proteins from proteins retained in the cytoplasm,using a method such as that provided in Example 12.

Host Cells.

The inducible coexpression systems of the invention are designed toexpress multiple gene products; in certain embodiments of the invention,the gene products are coexpressed in a host cell. Examples of host cellsare provided that allow for the efficient and cost-effective induciblecoexpression of components of multimeric products. Host cells caninclude, in addition to isolated cells in culture, cells that are partof a multicellular organism, or cells grown within a different organismor system of organisms. In addition, the expression constructs of theinducible coexpression systems of the invention can be used in cell-freesystems, such as those based on wheat germ extracts or on bacterial cellextracts, such as a continuous-exchange cell-free (CECF) proteinsynthesis system using E. coli extracts and an incubation apparatus suchas the RTS ProteoMaster (Roche Diagnostics GmbH; Mannheim, Germany) (Junet al., “Continuous-exchange cell-free protein synthesis usingPCR-generated DNA and an RNase E-deficient extract”, Biotechniques 2008March; 44(3): 387-391).

Prokaryotic Host Cells.

In some embodiments of the invention, expression constructs designed forcoexpression of gene products are provided in host cells, preferablyprokaryotic host cells. Prokaryotic host cells can include archaea (suchas Haloferax volcanii, Sulfolobus solfataricus), Grain-positive bacteria(such as Bacillus subtilis, Bacillus licheniformis, Brevibacilluschoshinensis, Lactobacillus brevis, Lactobacillus buchneri, Lactococcuslactis, and Streptomyces lividans), or Grain-negative bacteria,including Alphaproteobacteria (Agrobacterium tumefaciens, Caulobactercrescentus, Rhodobacter sphaeroides, and Sinorhizobium meliloti),Betaproteobacteria (Alcaligenes eutrophus), and Gammaproteobacteria(Acinetobacter calcoaceticus, Azotobacter vinelandii, Escherichia coli,Pseudomonas aeruginosa, and Pseudomonas putida). Preferred host cellsinclude Gammaproteobacteria of the family Enterobacteriaceae, such asEnterobacter, Erwinia, Escherichia (including E. coli), Klebsiella,Proteus, Salmonella (including Salmonella typhimurium), Serratia(including Serratia marcescans), and Shigella.

Eukaryotic Host Cells.

Many additional types of host cells can be used for the induciblecoexpression systems of the invention, including eukaryotic cells suchas yeast (Candida shehatae, Kluyveromyces lactis, Kluyveromycesfragilis, other Kluyveromyces species, Pichia pastoris, Saccharomycescerevisiae, Saccharomyces pastorianus also known as Saccharomycescarlsbergensis, Schizosaccharomyces pombe, Dekkera/Brettanomycesspecies, and Yarrowia lipolytica); other fungi (Aspergillus nidulans,Aspergillus niger, Neurospora crassa, Penicillium, Tolypocladium,Trichoderma reesia); insect cell lines (Drosophila melanogasterSchneider 2 cells and Spodoptera frugiperda Sf9 cells); and mammaliancell lines including immortalized cell lines (Chinese hamster ovary(CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidneycells (COS), human embryonic kidney (HEK, 293, or HEK-293) cells, andhuman hepatocellular carcinoma cells (Hep G2)). The above host cells areavailable from the American Type Culture Collection.

Alterations to Host Cell Gene Functions.

Certain alterations can be made to the gene functions of host cellscomprising inducible expression constructs, to promote efficient andhomogeneous induction of the host cell population by an inducer.Preferably, the combination of expression constructs, host cellgenotype, and induction conditions results in at least 75% (morepreferably at least 85%, and most preferably, at least 95%) of the cellsin the culture expressing gene product from each induced promoter, asmeasured by the method of Khlebnikov et al. described in Example 8. Forhost cells other than E. coli, these alterations can involve thefunction of genes that are structurally similar to an E. coli gene, orgenes that carry out a function within the host cell similar to that ofthe E. coli gene. Alterations to host cell gene functions includeeliminating or reducing gene function by deleting the geneprotein-coding sequence in its entirety, or deleting a large enoughportion of the gene, inserting sequence into the gene, or otherwisealtering the gene sequence so that a reduced level of functional geneproduct is made from that gene. Alterations to host cell gene functionsalso include increasing gene function by, for example, altering thenative promoter to create a stronger promoter that directs a higherlevel of transcription of the gene, or introducing a missense mutationinto the protein-coding sequence that results in a more highly activegene product. Alterations to host cell gene functions include alteringgene function in any way, including for example, altering a nativeinducible promoter to create a promoter that is constitutivelyactivated. In addition to alterations in gene functions for thetransport and metabolism of inducers, as described herein with relationto inducible promoters, and an altered expression of chaperone proteins,it is also possible to alter the carbon catabolite repression (CCR)regulatory system and/or the reduction-oxidation environment of the hostcell.

Carbon Catabolite Repression (CCR).

The presence of an active CCR regulatory system within a host can affectthe ability of an inducer to activate transcription from an induciblepromoter. For example, when a host cell such as E. coli is grown in amedium containing glucose, genes needed for the utilization of othercarbon sources, such as the araBAD and prpBCDE operons, are expressed ata low level if at all, even if the arabinose or propionate inducer isalso present in the growth medium. There is also a hierarchy ofutilization of carbon sources other than glucose: as in the case of theara and prp inducible promoter systems, where the presence of arabinosereduces the ability of propionate to induce expression from the prpBCDEpromoter (Park et al., “The mechanism of sugar-mediated cataboliterepression of the propionate catabolic genes in Escherichia coli”, Gene2012 Aug. 1; 504(1): 116-121; Epub 2012 May 3). The CCR mechanism of thecell therefore makes it more difficult to use two or more carbon-sourceinducers in an inducible coexpression system, as the presence of theinducer that is the preferred carbon source will inhibit induction byless-preferred carbon sources. The Park et al. authors attempted torelieve the repression of the prp promoter by arabinose, by using eithera mutant crp gene that produces an altered cAMP receptor protein thatcan function independently of cAMP, or a deletion of PTS(phosphotransferase system) genes involved in the regulation of CCR;both approaches were largely unsuccessful. However, the PTS-knockoutstrain used by the Park et al. authors is based on strain TP2811, whichis a deletion of the E. coli ptsHI-crr operon (Hernandez-Montalvo etal., “Characterization of sugar mixtures utilization by an Escherichiacoli mutant devoid of the phosphotransferase system”, Appl MicrobiolBiotechnol 2001 October; 57(1-2): 186-191). Deletion of the entireptsHI-crr operon has been found to affect total cAMP synthesis moresignificantly than a deletion of just the crr gene (Levy et al., “CyclicAMP synthesis in Escherichia coli strains bearing known deletions in thepts phosphotransferase operon”, Gene 1990 Jan. 31; 86(1): 27-33). Adifferent approach is to eliminate or reduce the function of ptsG genein the host cell, which encodes glucose-specific EII A (EII A^(glc)), akey element for CCR in E. coli (Kim et al., “Simultaneous consumption ofpentose and hexose sugars: an optimal microbial phenotype for efficientfermentation of lignocellulosic biomass”, Appl Microbiol Biotechnol 2010November; 88(5): 1077-1085, Epub 2010 Sep. 14). Another alteration inthe genome of a host cell such as E. coli, which leads to increasedtranscription of the prp promoter, is to eliminate or reduce the genefunction of the ascG gene, which encodes AscG. AscG is the repressor ofthe beta-D-glucoside-utilization operon ascFB under normal growthconditions, and also represses transcription of the prp promoter;disruption of the AscG coding sequence has been shown to increasetranscription from the prp promoter (Ishida et al., “Participation ofregulator AscG of the beta-glucoside utilization operon in regulation ofthe propionate catabolism operon”, J Bacteriol 2009 October; 191(19):6136-6144; Epub 2009 Jul. 24). A further alternative is to increaseexpression of the transcriptional regulator of promoters inducible bythe less-preferred carbon-source inducer, by placing it either under thecontrol of a strong constitutive promoter, or under the control of themore-preferred carbon-source inducer. For example, to increase theinduction of genes needed for the utilization of the less-preferredcarbon source xylose in the presence of the more-preferred arabinose,the coding sequence for XylR is placed into the E. coli araBAD operon(Groff et al., “Supplementation of intracellular XylR leads tocoutilization of hemicellulose sugars”, Appl Environ Microbiol 2012April; 78(7): 2221-2229, Epub 2012 Jan. 27). Host cells comprisinginducible coexpression constructs therefore preferably include anincreased level of gene function for transcriptional regulators ofpromoters inducible by the less-preferred carbon-source inducer(s), andan eliminated or reduced gene function for genes involved in the CCRsystem, such as crr and/or ptsG and/or ascG.

Cellular Transport of Heme and Other Cofactors.

When using the inducible coexpression systems of the invention toproduce enzymes that require cofactors for function, it is helpful touse a host cell capable of synthesizing the cofactor from availableprecursors, or taking it up from the environment. Common cofactorsinclude ATP, coenzyme A, flavin adenine dinucleotide (FAD), NAD⁺/NADH,and heme. Heine groups comprise an iron ion in the center of a largeheterocyclic organic ring called a porphyrin. The most common type ofheme group is heme B; other major types are heme A, heme C, and heme O,which vary in the side chains of the porphyrin. Hemin is a chloride saltof heme B and can be added to bacterial growth medium as a source ofheme. Other potential sources of heme include cytochromes, hemoglobin,and hemoglobin-containing substances such as blood. Laboratory strainsof E. coli derived from E. coli K12 typically lack an outer-membraneheme receptor, and thus do not transport heme into the cell, failing togrow in media where heme is the only iron source. Pathogenic strains ofE. coli such as O157:H7 and CFT073 contain an approximately 9-kb genomicsegment that is not present in E. coli K12, and that contains twodivergently transcribed operons encoding proteins involved in hemeuptake and utilization: the chuAS operon, and the chuTWXYUhmuV operon.This genomic segment is found in E. coli CFT073 and includes the NCBIReference Sequence NC 004431.1 (20 Jan. 2012) from position 4,084,974 to4,093,975. Transformation with the chuA gene (for example, NCBI Gene IDNo. 1037196), which encodes an outer-membrane hemin-specific receptor,was sufficient to confer on a K12-derived E. coli strain the ability togrow on hemin as an iron source (Torres and Payne, “Haem iron-transportsystem in enterohaemorrhagic Escherichia coli O157:H7”, Mol Microbiol1997 February; 23(4): 825-833). In addition to ChuA, some otherheterologous heme receptors can allow E. coli K12-derived strains totake up heme: Yersinia enterocolitica HemR, Serratia marcescens HasR,and Shigella dysenteria ShuA; and from grain-negative bacteria:Bordatella pertussis and B. bronchiseptica BhuR, Pseudomonas aeruginosaPhuR, and P. fluorescens PfhR. The ChuS protein is also involved in theutilization of heme: it is a heme-degrading heme oxygenase. In an E.coli aroB strain, which is deficient in the synthesis of theiron-chelating molecule enterobactin, transformation with chuS wasuseful in reducing cellular toxicity caused by growth on hemin in theabsence of enterobactin. Transcription of the chuAS and chuTWXYUhmuVoperons, and several other operons that are involved in iron metabolismand present in E. coli K12 strains, is repressed by the E. coli Furtranscriptional regulator when Fur is associated with Fe²⁺;transcription of these genes is thus activated when there is a drop inthe intracellular concentration of iron ions.

Host cells such as E. coli K12-derived strains can be altered to enablethem to take up heme by transforming them with all or part of thechuAS—chuTWXYUhmuV region containing at least chuA, or with the chuASoperon, optionally including additional genes from the chuTWXYUhmuVoperon. In embodiments where the promoter directing chuA transcriptionis repressible by Fur, Fur repression can be eliminated or reduced bydeleting the host cell gene encoding Fur, by growing host cells in theabsence of free iron, by growing host cells in the presence of achelator of free iron ions such as EDTA (ethylenediaminetetraaceticacid), or by transforming cells with polynucleotide constructs (such asplasmids maintained at high copy number) comprising multiple copies ofthe Fur binding site, to reduce the amount of Fur-Fe²⁺ complex availablefor repression of iron-metabolism operons. In a preferred embodiment, anexpression construct is introduced into the host cell, wherein theexpression construct comprises a polynucleotide encoding ChuA, andoptionally also encoding ChuS, under the transcriptional control of aconstitutive promoter.

Host Cell Reduction-Oxidation Environment.

Many multimeric gene products, such as antibodies, contain disulfidebonds. The cytoplasm of E. coli and many other cells is normallymaintained in a reduced state by the thioredoxin and theglutaredoxin/glutathione enzyme systems. This precludes the formation ofdisulfide bonds in the cytoplasm, and proteins that need disulfide bondsare exported into the periplasm where disulfide bond formation andisomerization is catalyzed by the Dsb system, comprising DsbABCD andDsbG. Increased expression of the cysteine oxidase DsbA, the disulfideisomerase DsbC, or combinations of the Dsb proteins, which are allnormally transported into the periplasm, has been utilized in theexpression of heterologous proteins that require disulfide bonds (Makinoet al., “Strain engineering for improved expression of recombinantproteins in bacteria”, Microb Cell Fact 2011 May 14; 10: 32). It is alsopossible to express cytoplasmic forms of these Dsb proteins, such as acytoplasmic version of DsbC (‘cDsbC’), that lacks a signal peptide andtherefore is not transported into the periplasm. Cytoplasmic Dsbproteins such as cDsbC are useful for making the cytoplasm of the hostcell more oxidizing and thus more conducive to the formation ofdisulfide bonds in heterologous proteins produced in the cytoplasm. Thehost cell cytoplasm can also be made more oxidizing by altering thethioredoxin and the glutaredoxin/glutathione enzyme systems directly:mutant strains defective in glutathione reductase (gor) or glutathionesynthetase (gshB), together with thioredoxin reductase (trxB), renderthe cytoplasm oxidizing. These strains are unable to reduceribonucleotides and therefore cannot grow in the absence of exogenousreductant, such as dithiothreitol (DTT). Suppressor mutations (ahpC*) inthe gene ahpC, which encodes the peroxiredoxin AhpC, convert it to adisulfide reductase that generates reduced glutathione, allowing thechanneling of electrons onto the enzyme ribonucleotide reductase andenabling the cells defective in gor and trxB, or defective in gshB andtrxB, to grow in the absence of DTT. A different class of mutated formsof AhpC can allow strains, defective in the activity ofgamma-glutamylcysteine synthetase (gshA) and defective in trxB, to growin the absence of DTT; these include AhpC V164G, AhpC S71F, AhpCE173/S71F, AhpC E171Ter, and AhpC dup162-169 (Faulkner et al.,“Functional plasticity of a peroxidase allows evolution of diversedisulfide-reducing pathways”, Proc Natl Acad Sci USA 2008 May 6;105(18): 6735-6740, Epub 2008 May 2). In such strains with oxidizingcytoplasm, exposed protein cysteines become readily oxidized in aprocess that is catalyzed by thioredoxins, in a reversal of theirphysiological function, resulting in the formation of disulfide bonds.

Another alteration that can be made to host cells is to express thesulfhydryl oxidase Erv1p from the inner membrane space of yeastmitochondria in the host cell cytoplasm, which has been shown toincrease the production of a variety of complex, disulfide-bondedproteins of eukaryotic origin in the cytoplasm of E. coli, even in theabsence of mutations in gor or trxB (Nguyen et al., “Pre-expression of asulfhydryl oxidase significantly increases the yields of eukaryoticdisulfide bond containing proteins expressed in the cytoplasm of E.coli” Microb Cell Fact 2011 Jan. 7; 10: 1). Host cells comprisinginducible coexpression constructs preferably also express cDsbC and/orErv1p, are deficient in trxB gene function, are also deficient in thegene function of either gor, gshB, or gshA, and express an appropriatemutant form of AhpC so that the host cells can be grown in the absenceof DTT.

Glycosylation of Polypeptide Gene Products.

Host cells can have alterations in their ability to glycosylatepolypeptides. For example, eukaryotic host cells can have eliminated orreduced gene function in glycosyltransferase and/oroligo-saccharyltransferase genes, impairing the normal eukaryoticglycosylation of polypeptides to form glycoproteins. Prokaryotic hostcells such as E. coli, which do not normally glycosylate polypeptides,can be altered to express a set of eukaryotic and prokaryotic genes thatprovide a glycosylation function (DeLisa et al., “Glycosylated proteinexpression in prokaryotes”, WO2009089154A2, 2009 Jul. 16).

Available Host Cell Strains with Altered Gene Functions.

To create preferred strains of host cells to be used in the induciblecoexpression systems and methods of the invention, it is useful to startwith a strain that already comprises desired genetic alterations (Table3).

TABLE 3 Host Cell Strains Strain: Genotype: Source: E. coli F− mcrAΔ(mrr-hsdRMS-mcrBC) Invitrogen Life Technologies TOP10 φ80lacZΔM15ΔlacX74 recA1 araD139 Catalog nos. C4040-10, Δ(ara-leu)7697 galU galKrpsL (Str^(R)) endA1 C4040-03, C4040-06, C4040- nupG λ- 50, and C4040-52E. coli Δ(ara-leu)7697 ΔlacX74 ΔphoA PvuII phoR Merck (EMD MilliporeOrigami ™ 2 araD139 ahpC galE galK rpsL F′[lac⁺ lacI^(q) Chemicals)Catalog No. 71344 pro] gor522::Tn10 trxB (Str^(R), Tet^(R)) E. colifhuA2 [lon] ompT ahpC gal λatt::pNEB3-r1- New England Biolabs CatalogSHuffle ® cDsbC (Spec, lacI) ΔtrxB sulA11 R(mcr- No. C3028H Express73::miniTn10--Tet^(S))2 [dcm] R(zgb-210::Tn10-- Tet^(S)) endA1 ΔgorΔ(mcrC-mrr)114::IS10

Methods of Altering Host Cell Gene Functions.

There are many methods known in the art for making alterations to hostcell genes in order to eliminate, reduce, or change gene function.Methods of making targeted disruptions of genes in host cells such as E.coli and other prokaryotes have been described (Muyrers et al., “Rapidmodification of bacterial artificial chromosomes by ET-recombination”,Nucleic Acids Res 1999 Mar. 15; 27(6): 1555-1557; Datsenko and Wanner,“One-step inactivation of chromosomal genes in Escherichia coli K-12using PCR products”, Proc Natl Acad Sci USA 2000 Jun. 6; 97(12):6640-6645), and kits for using similar Red/ET recombination methods arecommercially available (for example, the Quick & Easy E. coli GeneDeletion Kit from Gene Bridges GmbH, Heidelberg, Germany). In oneembodiment of the invention, the function of one or more genes of hostcells is eliminated or reduced by identifying a nucleotide sequencewithin the coding sequence of the gene to be disrupted, such as one ofthe E. coli K-12 substrain MG1655 coding sequences incorporated hereinby reference to the genomic location of the sequence, and morespecifically by selecting two adjacent stretches of 50 nucleotides eachwithin that coding sequence. The Quick & Easy E. coli Gene Deletion Kitis then used according to the manufacturer's instructions to insert apolynucleotide construct containing a selectable marker between theselected adjacent stretches of coding sequence, eliminating or reducingthe normal function of the gene. Red/ET recombination methods can alsobe used to replace a promoter sequence with that of a differentpromoter, such as a constitutive promoter, or an artificial promoterthat is predicted to promote a certain level of transcription (De Mey etal., “Promoter knock-in: a novel rational method for the fine tuning ofgenes”, BMC Biotechnol 2010 Mar. 24; 10: 26). The function of host cellgenes can also be eliminated or reduced by RNA silencing methods (Man etal., “Artificial trans-encoded small non-coding RNAs specificallysilence the selected gene expression in bacteria”, Nucleic Acids Res2011 April; 39(8): e50, Epub 2011 Feb. 3). Further, known mutations thatalter host cell gene function can be introduced into host cells throughtraditional genetic methods.

Inducible Coexpression Systems of the Invention

Inducible coexpression systems of the invention involve host cellscomprising two or more expression constructs, where the expressionconstructs comprise inducible promoters directing the expression of geneproducts, and the host cells have altered gene functions that allow forhomogeneous inducible expression of the gene products. FIG. 1 shows aschematic representation of an inducible coexpression system of theinvention, with the following components: (1) host cell, (2) host genome(including genetic alterations), (3) an expression vector ‘X’ comprisingan inducible promoter directing expression of a gene product, (4) adifferent expression vector ‘Y’ comprising an inducible promoterdirecting expression of another gene product, (5) chemical inducers ofexpression, and (6) the multimeric coexpression product.

FIG. 2 shows a schematic representation of a particular example of aninducible coexpression system of the invention, utilizing the araBADpromoter on a pBAD24 expression vector in combination with apropionate-inducible promoter (prpBCDE promoter) on a pPRO33 expressionvector (U.S. Pat. No. 8,178,338 B2; May 15, 2012; Keasling, Jay), in anE. coli host cell housing the appropriate genomic alterations whichallow for homogenously inducible expression. In this manner, tightcontrol and optimization of expression of each component of a multimericproduct can be achieved for use in a number of coexpressionapplications. In this embodiment, the host cell (1) is theGrain-negative bacterium Escherichia coli, commonly used in the art forprotein expression. The host genome (2) is the genome of the host cellorganism with mutations or other alterations that facilitatehomogenously inducible protein coexpression, including expression of acytoplasmic form of the disulfide isomerase DsbC which lacks a signalpeptide. In one embodiment, the genomic alterations include both anaraBAD operon knockout mutation, and either expression of araE andaraFGH from constitutive promoters, or a point mutation in the lacY gene(A117C) in an araEFGH-deficient background, to facilitate homogenousinduction of plasmid-based ara promoters with exogenously appliedL-arabinose, and also an inactivated proprionate metabolism gene, prpD,to facilitate homogenous induction of plasmid-based propionate promoterswith exogenously applied propionate, which is converted to2-methylcitrate in vivo. Other genomic alterations that are useful forthe inducible coexpression system, and may be introduced into the hostcell, include without limitation: targeted inactivation of thescpA-argK-scpBC operon, to reduce background expression from the prpBCDEpromoter; expression of the transcriptional regulator (prpR) for theless-preferred carbon-source (propionate) from an L-arabinose-induciblepromoter such as the araBAD promoter, and/or an eliminated or reducedgene function for genes involved in the CCR system, such as crr and/orptsG, to avoid suppression by the CCR system of induction by propionatein the presence of L-arabinose; reductions in the level of gene functionfor glutathione reductase (gor) or glutathione synthetase (gshB),together with thioredoxin reductase (trxB), and/or expression of yeastmitochondrial sulfhydryl oxidase Erv1p in the host cell cytoplasm, toprovide a less strongly reducing environment in the host cell cytoplasmand promote disulfide bond formation; increased levels of expression,such as from a strong constitutive promoter, of chaperone proteins suchas DnaK/DnaJ/GrpE, DsbC/DsbG, GroEL/GroES, IbpA/IbpB, Skp, Tig (triggerfactor), and/or FkpA; and other mutations to reduce endogenous proteaseactivity (such as that of the Lon and OmpT proteases) and recombinaseactivities.

As shown in FIG. 2, two compatible expression vectors (3, 4) aremaintained in the host cell to allow for simultaneous expression(coexpression) of two different gene products. In this embodiment, oneexpression vector (′L-arabinose-induced expression vector′) contains anL-arabinose-induced promoter, and is similar or identical to pBAD orrelated plasmids in which an araBAD promoter drives expression of aninserted expression sequence cloned into the multiple cloning site(MCS). The L-arabinose-induced expression vector also contains a codingsequence for an antibiotic-resistance gene (such as the Tn3 bla gene,which encodes beta-lactamase and confers resistance to ampicillin) tofacilitate selection of host cells (bacterial colonies) which contain anintact expression vector. An origin of replication (ORI) is required forpropagation of the plasmid within bacterial host cells. The L-arabinoseinduced expression plasmid also contains a polynucleotide sequenceencoding araC, a transcriptional regulator that allows for L-arabinoseinduction of the araBAD promotor and through transcriptional repressionreduces ‘leaky’ background expression in the non-induced state. Theother expression vector (‘propionate-induced expression vector’) issimilar or identical to pPRO or related plasmids, in which apropionate-induced promoter drives expression of an inserted expressionsequence cloned into the multiple cloning site (MCS). The plasmid alsocontains a coding sequence for an antibiotic-resistance gene (such asthe cat gene, encoding chloramphenicol acetyltransferase, which confersresistance to chloramphenicol) to facilitate selection of host cellswhich contain an intact expression vector. An origin of replication(ORI) is required for propagation of the plasmid within bacterial hostcells. In addition, the propionate-induced expression vector contains apolynucleotide sequence encoding prpR, a transcriptional regulator thatallows for propionate (2-methylcitrate) induction of the prpBCDEpromoter and reduces ‘leaky’ background expression in the non-inducedstate. To facilitate separate titratation of induction, plasmidcompatibility, and copropagation of the expression vectors, it is usefulfor the expression vectors to contain promoters responsive to differentinducers, compatible origins of replication, and differentantibiotic-resistance markers. In one embodiment of the invention, apBAD24 or related expression vector (pMB1 or ‘pBR322’ ORI, Amp^(R))containing an L-arabinose-inducible araBAD promoter is combined in ahost cell with a pPRO33 or related expression vector (p15A ORI, Cm^(R))containing a propionate-inducible prpBCDE promoter. The expressionvectors are co-propagated and maintained using growth mediumsupplemented with ampicillin and chloramphenicol. In one embodiment, oneexpression vector comprises a polynucleotide sequence encoding the heavychain of a full-length antibody, and the other expression vectorcomprises a polynucleotide sequence encoding the light chain of afull-length antibody, each coding sequence cloned in-frame into the MCSof the respective expression vector. For production of certain geneproducts such as antibodies, coding sequence optimization for the hostorganism (including adjustment for codon bias and GC-content, amongother considerations) will determine the coding sequences to be insertedinto the expression constructs of the coexpression system.

Referring again to FIG. 2, coexpression of gene products is induced byinexpensive exogenously applied chemical metabolites, L-arabinose andpropionate (5). The level of induction of expression of each geneproduct is independently titrated with its own chemical inducer, therebyfacilitating optimization of protein coexpression. This is useful forexpression of protein complexes and proteins that require a bindingpartner for stabilization, and may facilitate expression of otherwisedifficult to express proteins, such as those with poor solubility orcellular toxicity. In this example, upon induction, antibody heavy andshort chains are each separately expressed, then the proteins join andform interchain disulfide bridges (within the cytoplasm of the bacterialhost) which allows the formation and stabilization of full-lengthantibody comprised of the heavy and light chains. Proteins can bedirected to various compartments of the host organism. For example, inE. coli the protein can be expressed in the cytoplasm, cell membrane,periplasm, or secreted into the medium. After an appropriate incubationtime, cells and media are collected, and the total protein extracted,which includes the coexpressed gene products (6). After extraction, thedesired product can be purified using a number of methods well known inthe art depending on the nature of the gene products produced in thecoexpression system (for example liquid chromatography). In the exampleshown in FIG. 2, the multimeric product (full-length antibody) isextracted and purified using chromatographic methods. Purified intactantibody is visualized on a non-denaturing gel using standardtechniques, including protein-binding dyes or immunohistochemistry. Thefull-length antibody product can then be used for a number of research,diagnostic, or other applications.

Products Made by the Methods of the Invention

There is broad versatility in utilizing the inducible coexpressionsystems of the present invention in numerous coexpression applications,and in the properties of the products.

Glycosylation.

Gene products coexpressed by the methods of the invention may beglycosylated or unglycosylated. In one embodiment of the invention, thecoexpressed gene products are polypeptides. Glycosylated polypeptidesare polypeptides that comprise a covalently attached glycosyl group, andinclude polypeptides comprising all the glycosyl groups normallyattached to particular residues of that polypeptide (fully glycosylatedpolypeptides), partially glycosylated polypeptides, polypeptides withglycosylation at one or more residues where glycosylation does notnormally occur (altered glycosylation), and polypeptides glycosylatedwith at least one glycosyl group that differs in structure from theglycosyl group normally attached to one or more specified residues(modified glycosylation). An example of modified glycosylation is theproduction of “defucosylated” or “fucose-deficient” polypeptides,polypeptides lacking fucosyl moieties in the glycosyl groups attached tothem, by expression of polypeptides in host cells lacking the ability tofucosylate polypeptides. Unglycosylated polypeptides are polypeptidesthat do not comprise a covalently bound glycosyl group. Anunglycosylated polypeptide can be the result of deglycosylation of apolypeptide, or of production of an aglycosylated polypeptide.Deglycosylated polypeptides can be obtained by enzymaticallydeglycosylating glycosylated polypeptides, whereas aglycosylatedpolypeptides can be produced by expressing polypeptides in host cellsthat do not have the capability to glycosylate polypeptides, such asprokaryotic cells or cells in which the function of at least oneglycosylation enzyme has been eliminated or reduced. In a particularembodiment, the coexpressed polypeptides are aglycosylated, and in amore specific embodiment, the aglycosylated polypeptides are coexpressedin prokaryotic cells such as E. coli.

Other Modifications of Gene Products.

Gene products coexpressed by the methods of the invention may becovalently linked to other types of molecules. Examples of moleculesthat may be covalently linked to coexpressed gene products, withoutlimiting the scope of the invention, include polypeptides (such asreceptors, ligands, cytokines, growth factors, polypeptide hormones,DNA-binding domains, protein interaction domains such as PDZ domains,kinase domains, antibodies, and fragments of any such polypeptides);water-soluble polymers (such as polyethylene glycol (PEG),carboxymethylcellulose, dextran, polyvinyl alcohol, polyoxyethylatedpolyols (such as glycerol), polyethylene glycol propionaldehyde, andsimilar compounds, derivatives, or mixtures thereof); and cytotoxicagents (such as chemotherapeutic agents, growth-inhibitory agents,toxins (such as enzymatically active toxins of bacterial, fungal, plant,or animal origin, or fragments thereof), and radioactive isotopes).

In addition, gene products to be coexpressed by the methods of theinvention can be designed to include molecular moieties that aid in thepurification and/or detection of the gene products. Many such moietiesare known in the art; as one example, a polypeptide gene product can bedesigned to include a polyhistidine ‘tag’ sequence—a run of six or morehistidines, preferably six to ten histidine residues, and mostpreferably six histidines—at its N- or C-terminus. The presence of apolyhistidine sequence on the end of a polypeptide allows it to be boundby cobalt- or nickel-based affinity media, and separated from otherpolypeptides. The polyhistidine tag sequence can be removed byexopeptidases. As another example, fluorescent protein sequences can beexpressed as part of a polypeptide gene product, with the amino acidsequence for the fluorescent protein preferably added at the N- orC-terminal end of the amino acid sequence of the polypeptide geneproduct. The resulting fusion protein fluoresces when exposed to lightof certain wavelengths, allowing the presence of the fusion protein tobe detected visually. A well-known fluorescent protein is the greenfluorescent protein of Aequorea victoria, and many other fluorescentproteins are commercially available, along with nucleotide sequencesencoding them.

Antibodies.

In one embodiment of the invention, the coexpressed gene products areantibodies. The term ‘antibody’ is used in the broadest sense andspecifically includes ‘native’ antibodies, fully-human antibodies,humanized antibodies, chimeric antibodies, multispecific antibodies(such as bispecific antibodies), monoclonal antibodies, polyclonalantibodies, antibody fragments, and other polypeptides derived fromantibodies that are capable of binding antigen. Unless indicatedotherwise herein, the numbering of the residues in an immunoglobulinheavy chain (‘EU numbering’) is that of the EU index (the residuenumbering of the human IgG1 EU antibody) as in Kabat et al., Sequencesof Proteins of Immunological Interest, Fifth Edition, 1991, NationalInstitute of Health, Bethesda, Md.

‘Native’ antibodies are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of inter-chaindisulfide linkages varies among the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at itsN-terminal end a variable domain (V_(H)) followed by a number ofconstant domains. Each light chain has a variable domain at itN-terminal end (V_(L)) and a constant domain at its C-terminal end; theconstant domain of the light chain is aligned with the first constantdomain of the heavy chain, and the light-chain variable domain isaligned with the variable domain of the heavy chain. The term ‘variable’refers to the fact that certain portions of the variable domains differextensively in sequence among antibodies and are used in the binding andspecificity of each particular antibody for an antigen. However, thevariability is not evenly distributed throughout the variable domains ofantibodies. It is concentrated in three segments called hypervariableregions (HVRs) both in the light-chain and the heavy-chain variabledomains. The more highly conserved portions of variable domains arecalled the framework regions (FR). The variable domains of native heavyand light chains each comprise four FR regions, connected by three HVRs,and with the HVRs from the other chain, contribute to the formation ofthe antigen-binding site of antibodies.

The term ‘Fc region’ refers to a C-terminal region of an immunoglobulinheavy chain, and includes native Fc regions and variant Fc regions.Although the boundaries of the Fc region of an immunoglobulin heavychain might vary, the human IgG heavy-chain Fc region can be defined tostretch from an amino acid residue at position Cys226, or from Pro230,to the carboxyl-terminus thereof. Alternatively, the Fc region can bedefined to extend from the N-terminal residue (Ala231) of the conservedC_(H)2 immunoglobulin domain to the C-terminus, and may include multipleconserved domains such as C_(H)2, C_(H)3, and C_(H)4. The C-terminallysine (residue 447 according to the EU numbering system) of the nativeFc region may be removed, for example, during production or purificationof the antibody, or by recombinantly engineering the nucleic acidencoding a heavy chain of the antibody. Accordingly, a composition ofintact antibodies may comprise antibody populations with all K447residues removed, antibody populations with no K447 residues removed,and antibody populations having a mixture of antibodies with and withoutthe K447 residue. The Fc region of an antibody is crucial forrecruitment of immunological cells and antibody dependent cytotoxicity(ADCC). In particular, the nature of the ADCC response elicited byantibodies depends on the interaction of the Fc region with receptors(FcRs) located on the surface of many cell types. Humans contain atleast five different classes of Fc receptors. The binding of an antibodyto FcRs determines its ability to recruit other immunological cells andthe type of cell recruited. Hence, the ability to engineer antibodieswith altered Fc regions that can recruit only certain kinds of cells canbe critically important for therapy (US Patent Application 20090136936A1, 05-28-2009, Georgiou, George). Native antibodies produced bymammalian cells typically comprise a branched, biantennaryoligosaccharide that is generally attached by an N-linkage to Asn297 ofthe CH2 domain of the Fc region. In certain embodiments, antibodiesproduced by the methods of the invention are not glycosylated or areaglycosylated, for example, due to a substitution at residue 297 of theFc region, or to expression in a host cell that does not have thecapability to glycosylate polypeptides. Due to altered ADCC responses,unglycosylated antibodies may stimulate a lower level of inflammatoryresponses such as neuroinflammation. Also, since an antibody having anaglycosylated Fc region has very low binding affinity for Fc receptors,such antibodies would not bind to the large number of immune cells thatbear these receptors. This is a significant advantage since it reducesnon-specific binding, and also increases the half-life of the antibodyin vivo, making this attribute very beneficial in therapeutics.

The terms ‘full-length antibody’, ‘intact antibody’, and ‘wholeantibody’ are used interchangeably to refer to an antibody in itssubstantially intact ‘native’ form, not antibody fragments as definedbelow. The terms particularly refer to an antibody with heavy chainsthat each comprise a variable domain and an Fc region. ‘Antibodyfragments’ comprise a portion of an intact antibody, preferablycomprising the antigen-binding region thereof. Examples of antibodyfragments include Fab, Fab′, F(ab′)₂, Fc, Fd, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules such asscFv; and multispecific antibodies formed from antibody fragments.

A ‘human antibody’ is one that possesses an amino-acid sequencecorresponding to that of an antibody produced by a human. A ‘chimeric’antibody is one in which a portion of the heavy and/or light chain isidentical to, or shares a certain degree of amino acid sequence identitywith, corresponding sequences in antibodies derived from a particularspecies or belonging to a particular antibody class or subclass, whilethe remainder of the chain(s) is identical to, or shares a certaindegree of amino acid sequence identity with, corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies. A‘humanized’ antibody is a chimeric antibody that contains minimal aminoacid residues derived from non-human immunoglobulin molecules. In oneembodiment, a humanized antibody is a human immunoglobulin (recipientantibody) in which HVR residues of the recipient antibody are replacedby residues from an immunoglobulin HVR of a non-human species (donorantibody) such as mouse, rat, rabbit, or nonhuman primate. In someinstances, FR residues of the human recipient antibody are replaced bycorresponding non-human residues. Furthermore, humanized antibodies maycomprise residues that are not found in the recipient antibody or in thedonor antibody. The term ‘monoclonal antibody’ refers to an antibodyobtained from a population of substantially homogeneous antibodies, inthat the individual antibodies comprising the population are identicalexcept for possible mutations, such as naturally occurring mutations,that may be present in minor amounts. Thus, the modifier ‘monoclonal’indicates the character of the antibody as not being a mixture ofdiscrete antibodies. In contrast to polyclonal antibody preparations,which typically include different antibodies directed against differentdeterminants (epitopes), each monoclonal antibody of a monoclonalantibody preparation is directed against the same single determinant onan antigen. In addition to their specificity, monoclonal antibodypreparations are advantageous in that they are typically uncontaminatedby other immunoglobulins.

The ‘binding affinity’ of a molecule such as an antibody generallyrefers to the strength of the sum total of non-covalent interactionsbetween a single binding site of a molecule and its binding partner(such as an antibody and the antigen it binds). Unless indicatedotherwise, ‘binding affinity’ refers to intrinsic binding affinity thatreflects a 1:1 interaction between members of a binding pair (such asantibody and antigen). The affinity of a molecule X for its partner Ycan generally be represented by the dissociation constant (Kd).Low-affinity antibodies (higher Kd) generally bind antigen slowly andtend to dissociate readily, whereas high-affinity antibodies (lower Kd)generally bind antigen faster and tend to remain bound longer. A varietyof ways to measure binding affinity are known in the art, any of whichcan be used for purposes of the present invention. Specific illustrativemethods for measuring binding affinity are described in Example 10.Antibodies and antibody fragments produced by and/or used in methods ofthe invention preferably have binding affinities of less than 100 nM,more preferably have binding affinities of less than 10 nM, and mostpreferably have binding affinities of less than 2 nM, as measured by asurface-plasmon resonance assay as described in Example 10.

Antibodies (secondary) that recognize aglycosylated antibodies.Production of antibodies in E. coli-based or other prokaryoticexpression systems without glycosylation enzymes will generally yieldaglycosylated antibodies, which can be used as primary antibodies. Inaddition to using the inducible coexpression systems of the invention toproduce aglycosylated primary antibodies, the inducible coexpressionsystems of the invention can also be used to efficiently producesecondary antibodies that specifically recognize aglycosylated primaryantibodies. One aspect of the present invention is a secondary antibodysystem capable of detecting an unglycosylated or aglycosylated primaryantibody for research, analytic, diagnostic, or therapeutic purposes. Asone example, a secondary antibody system is provided with the followingcomponents: epitope, primary antibody, secondary antibody, and detectionsystem. The epitope is a portion of an antigen (usually a protein) whichis the antigenic determinant that produces an immunological responsewhen introduced into a live animal or is otherwise recognizable by anantibody. In practice, the epitope of interest may be present within amixture or a tissue. In one embodiment, the epitope is a proteinexpressed in carcinoma cells in human tissue. The primary antibody is anantibody fragment, a single full-length antibody (monoclonal), or amixture of different full-length antibodies (polyclonal), whichrecognizes and binds to the epitope, and preferably binds specificallyto the epitope. A full-length antibody in this example comprises twoheavy polypeptide chains and two light polypeptide chains joined bydisulfide bridges. Each of the chains comprises a constant region (Fc)and a variable region (Fv). There are two antigen binding sites in thefull-length antibody. In one embodiment of the present invention, theprimary antibody is a full-length aglycosylated antibody (such as thatproduced in an E. coli-based expression system) which recognizes andbinds an epitope of interest. The secondary antibody is an antibodyfragment, a single full-length antibody (monoclonal), or a mixture ofdifferent full-length antibodies (polyclonal), which recognizes andbinds to the aglycosylated primary antibody, and preferably bindsspecifically to the aglycosylated primary antibody. In one embodiment ofthe present invention, the secondary antibody is a full-length antibodywhich recognizes and binds the aglycosylated Fc portion of a full-lengthprimary antibody. In this case, the antibody binding sites are selectedand/or engineered to specifically recognize the Fc portion of theaglycosylated primary antibody, with or without the C-terminal lysineresidue. In other embodiments, the secondary antibody could beengineered to recognize additional regions (epitopes) of theaglycosylated primary antibody, or additional engineered epitopesincluding but not limited to polypeptide sequences covalently attachedto the primary antibody. The secondary antibody can be directed atsingle or multiple sites (epitopes) present on full-length aglycosylatedantibodies molecules (including various immunoglobulin classes such asIgG, IgA, etc.) or antibody fragments such as Fc or Fab. Therefore, somesecondary antibodies generated in this way would have broad specificityfor any aglycosylated full-length antibody. The primary and secondaryantibodies of the present invention can also include those produced bytraditional methods (polyclonal antibody production using immunizedrabbits or monoclonal antibody production using mouse hybridomas) andrecombinant DNA technology such as phage display methods for identifyingantigen-binding polypeptides.

Detection systems generally comprise an agent that is linked to or whichbinds the secondary antibody, enabling detection, visualization, and/orquantification of the secondary antibody. Various detection systems arewell known in the art including but not limited to fluorescent dyes,enzymes, radioactive isotopes, or heavy metals. These may or may notinvolve direct physical linkage of additional polypeptides to thesecondary antibody. Applications of this secondary antibody systeminclude but are not limited to immunohistochemistry, Western blotting,and enzyme-linked immunosorbent assay (ELISA). For example, in oneembodiment for use in immunohistochemistry, the epitope of interestwould be present on a thin section of tissue, then an aglycosylatedprimary antibody would be applied to the tissue and allowed to bind theepitope. The unbound primary antibody would be removed, and then asecondary antibody capable of specifically binding the aglycosylatedprimary antibody is applied to the tissue and allowed to bind to theprimary antibody. The unbound secondary antibody would be removed, andthen detection system reagents applied. For example, if the secondaryantibody were linked to an enzyme, then colorigenic enzymatic substrateswould be applied to the tissue and allowed to react. Direct microscopicor fluoroscopic visualization of the reactive enzymatic substrates couldthen be performed. Other detection methods are well known in the art.The advantages of a system using secondary antibodies that recognizeaglycosylated antibodies include, without limitation, the following: 1)increased specificity in immunohistochemistry because the secondaryantibody is designed to bind the aglycosylated Fc portion of the primaryantibody which is not otherwise present in eukaryotic tissues; 2)decreased background staining because of increased specificity for theprimary antibody; 3) decreased cost of secondary antibody systemproduction because the primary and/or secondary antibodies can begenerated in prokaryotes such as E. coli; and 4) avoiding unnecessaryutilization of mammals, including mice and rabbits, because the entireprocess of antibody development can be performed in prokaryotes such asE. coli.

Enzymes Used in Industrial Applications.

Many industrial processes utilize enzymes that can be produced by themethods of the invention. These processes include treatment ofwastewater and other bioremediation and/or detoxification processes;bleaching of materials in the paper and textile industries; anddegradation of biomass into material that can be fermented efficientlyinto biofuels. In many instances it would be desirable to produceenzymes for these applications in microbial host cells or preferably inbacterial host cells, but the active enzyme is difficult to express inlarge quantities due to problems with enzyme folding and/or arequirement for a cofactor. In the following embodiments of theinvention, the inducible coexpression methods of the invention are usedto produce enzymes with inductrial applications.

Arabinose- and Xylose-Utilization Enzymes.

D-xylose is the most abundant pentose in plant biomass, found inpolysaccharides, hemicellulose, and pectin, with L-arabinose being thesecond most abundant pentose. For the development and production ofbiofuels and other bioproducts, it is useful to convert D-xylose andL-arabinose into hexoses including glucose and fructose, as hexoses aremore efficiently fermented into biofuels such as ethanol. As describedabove, the E. coli araBAD operon encodes proteins that metabolizeL-arabinose as follows: L-arabinose by L-arabinose isomerase (AraA, EC5.3.1.4) to L-ribulose; L-ribulose by L-ribulokinase (AraB, EC 2.7.1.16)to L-ribulose-phosphate; L-ribulose-phosphate by L-ribulose-5-phosphate4-epimerase (AraD, EC 5.1.3.4) to D-xylulose-5-phosphate (also calledD-xylulose-5-P), which is part of the pentose phosphate pathway to theformation of fructose and glocose. Another enzymatic pathway (an“oxo-reductive pathway”) that converts L-arabinose to xylitol, which canthen be converted to D-xylulose-5-P, is as follows: L-arabinose byL-arabinose/D-xylose reductase (EC 1.1.1.21) to L-arabinitol;L-arabinitol by L-arabinitol dehydrogenase (EC 1.1.1.12) to L-xylulose;and L-xylulose by L-xylulose reductase (EC 1.1.1.10) to xylitol. The E.coli xylAB operon encodes one enzymatic pathway (the “isomerasepathway”) for utilizing D-xylose, as follows: D-xylose by D-xyloseisomerase (XylA, EC 5.3.1.5) to D-xylulose; D-xylulose by xylulokinase(XylB, EC 2.7.1.17) to D-xylulose-5-P. Another enzymatic pathway (an“oxo-reductive pathway”) for converting D-xylose to D-xylulose-5-P is:D-xylose by D-xylose reductase (EC 1.1.1.21) to xylitol; xylitol byxylitol dehydrogenase (EC 1.1.1.9) to D-xylulose; D-xylulose byxylulokinase (XylB, EC 2.7.1.17) to D-xylulose-5-P. Because of thevarying cofactors needed in the oxo-reductive pathways, such as NADPH,NAD⁻, and ATP, and the degree to which these cofactors are available forusage, an imbalance can result in an overproduction of xylitolbyproduct. D-xylulose-5-P plus erythrose 4-phosphate can be converted bytransketolase (EC 2.2.1.1) to glyceraldehyde 3-phosphate plus fructose6-phosphate.

The inducible coexpression methods of the invention can be used toproduce arabinose- and xylose-utilization enzymes, which are defined asbeing the enzymes listed by EC number in the preceding paragraph. The EC(or ‘Enzyme Commission’) number for each enzyme is established by theInternational Union of Biochemistry and Molecular Biology (IUBMB).Further information about these enzymes and specific examples of themcan readily be obtained through the UniProt protein database(www.uniprot.org/uniprot) and the BRENDA database,(www.brenda-enzymes.org/index); the BRENDA and UniProt database entriesfor the arabinose- and xylose-utilization enzymes are incorporated byreference herein. In some embodiments, an arabinose- orxylose-utilization enzyme is coexpressed with a chaperone protein; infurther embodiments of the invention, an arabinose- orxylose-utilization enzyme is coexpressed with a transporter for acofactor. In certain embodiments, L-arabinose isomerase (AraA, EC5.3.1.4) or L-arabinose reductase (EC 1.1.1.21) is produced by themethods of the invention; in these embodiments, an arabinose-induciblepromoter is not utilized in any expression construct because theinducer, L-arabinose, would be converted to L-ribulose or L-arabinitol,respectively. For production of arabinose-utilization enzymes other thanL-arabinose isomerase (AraA, EC 5.3.1.4) or L-arabinose reductase (EC1.1.1.21), an arabinose-inducible promoter can be utilized if the hostcell is deficient in EC 5.3.1.4 and/or EC 1.1.1.21, such as an araAmutant, and cannot catabolize L-arabinose. Similarly, in embodimentswhere D-xylose isomerase (XylA, EC 5.3.1.5) or D-xylose reductase (EC1.1.1.21) is produced by the methods of the invention, axylose-inducible promoter is not utilized in any expression constructbecause the inducer, D-xylose, would be converted to D-xylulose orxylitol, respectively. For production of xylose-utilization enzymesother than D-xylose isomerase (XylA, EC 5.3.1.5) or D-xylose reductase(EC 1.1.1.21), a xylose-inducible promoter can be utilized if the hostcell is deficient in EC 5.3.1.5 and/or EC 1.1.1.21, such as a xylAmutant, and cannot catabolize D-xylose.

Xylose isomerase (XylA, EC 5.3.1.5) is an enzyme found inmicroorganisms, anaerobic fungi, and plants, which catalyzes theinterconversion of an aldo sugar (D-xylose) to a keto sugar(D-xylulose). It can also isomerize D-ribose to D-ribulose and D-glucoseto D-fructose. This enzyme belongs to the family of isomerases,specifically those intramolecular oxidoreductases interconvertingaldoses and ketoses. The systematic name of this enzyme class isD-xylose aldose-ketose-isomerase. Other names in common use includeD-xylose isomerase, D-xylose ketoisomerase, D-xylose ketol-isomerase,and glucose isomerase. The enzyme is used industrially to convertglucose to fructose in the manufacture of high-fructose corn syrup, andas described above can be used in the conversion of pentoses to hexosesfor biofuel production. Xylose isomerase is a homotetramer and requirestwo divalent cations—Mg²⁺, Mn²⁺, and/or Co²⁺—for maximal activity.Xylose isomerase activity can be measured using an NADH-linked arabitoldehydrogenase assay (Smith et al., “D-Xylose (D-glucose) isomerase fromArthrobacter strain N.R.R.L. B3728. Purification and properties”,Biochem J 1991 Jul. 1; 277 (Pt 1): 255-261), in which one unit of xyloseisomerase activity is the amount of enzyme that converts 1 micromol ofD-xylose into D-xylulose in one minute. In at least some species, xyloseisomerase requires magnesium (or manganese in the case of plants) forits activity, while cobalt may be necessary to stabilize the tetramericstructure of the enzyme. Each xylose isomerase subunit contains analpha/beta-barrel fold similar to that of other divalent metal-dependentTIM (triosephosphate isomerase) barrel enzymes, and the C-terminalsmaller part forms an extended helical fold implicated inmultimerization. Conserved residues in all known xylose isomerases are ahistidine in the N-terminal section of the enzyme, shown to be involvedin the catalytic mechanism of the enzyme, and two glutamate residues, ahistidine, and four aspartate residues that form the two metal-bindingsites, each of which binds an ion of magnesium, cobalt, or manganese(Katz et al., “Locating active-site hydrogen atoms in D-xyloseisomerase: time-of-flight neutron diffraction”, Proc Natl Acad Sci USA2006 May 30; 103(22): 8342-8347; Epub 2006 May 17).

In some embodiments of the invention, inducible coexpression is used toexpress a xylose isomerase protein; in certain embodiments, the xyloseisomerase (“XI”) is selected from the group consisting of: Arthrobactersp. strain NRRL B3728 XI (UniProt P12070); Bacteroides stercoris XI(UniProt BONPH3); Bifidobacterium longum XI (UniProt Q8G3Q1);Burkholderia cenocepacia XI (UniProt Q1BG90); Ciona intestinalis XI(UniProt F6WBF5); Clostridium phytofermentans XI (UniProt A9KN98);Orpinomyces sp. ukk1 XI (UniProt B7SLY1); Piromyces sp. E2 XI (UniProtQ9P8C9); Streptomyces lividans XI (UniProt Q9RFM4); Streptomyceslividans TK24 XI (UniProt D6ESI7); Thermoanaerobacter ethanolicus JW 200XI (UniProt D2DK62); Thermoanaerobacter yonseii XI (UniProt Q9KGU2);Thermotoga neapolitana XI (UniProt P45687); Thermus thermophilus XI(UniProt P26997); and Vibrio sp. strain XY-214 XI (UniProt C7G532). Inparticular embodiments, a xylose isomerase is inducibly coexpressed witha divalent ion transporter such as CorA (UniProt P0ABI4): using aninducible promoter to control the timing and extent of the transport ofions can be helpful in reducing toxicity to host cells from metal ionssuch as Co^(2±). In additional embodiments, mutations are introducedinto xylose isomerase proteins that affect the interaction between pairsof XI monomers; for example, the introduction of cysteine residues sothat disulfide bonds between a pair of monomers can be formed (seeVarsani et al., “Arthrobacter D-xylose isomerase: protein-engineeredsubunit interfaces”, Biochem J 1993 Apr. 15; 291 (Pt 2): 575-583). Inthis example, because cysteine residues are introduced in reciprocal butnot identical locations within the monomers, two different types ofaltered monomers are produced, and the inducible coexpression systems ofthe invention are used to titrate the relative expression of the twotypes of monomers to achieve the desired stoichiometric ratio.

Lignin-Degrading Peroxidases.

Peroxidases are a subgroup of oxidoreductases and are used to catalyze avariety of industrial processes. Oxidoreductases can break down ligninor act as reductases in the degradation of cellulose and hemi-cellulose,allowing the enzymes used in the processing of plant biomass to moreeasily access the saccharide residues during the production of biofuelssuch as ethanol. Oxidoreductases can be oxidases or dehydrogenases.Oxidases use molecular oxygen as an electron acceptor, whiledehydrogenases oxidize a substrate by transferring an H⁻ group to anacceptor, such as NAD/NADP⁺ or a flavin-dependent enzyme. Peroxidasescatalyze the reduction of a peroxide, such as hydrogen peroxide (H₂O₂).Other types of oxidoreductases include oxygenases, hydroxylases, andreductases. In addition to oxygen, flavin adenine dinucleotide (FAD),and the nicotinamide adenine dinucleotides NAD and NADP, potentialcofactors of oxidoreductases include cytochromes and heroes, disulfide,and iron-sulfur proteins.

Lignin-degrading peroxidases can oxidize a variety of aromatic compoundsincluding high-redox-potential compounds such as lignin, industrialdyes, pesticides, etc. Four types of lignin-modifying enzymes have beenidentified and characterized: lignin peroxidase (LiP, EC 1.11.1.14),manganese peroxidase (MnP, EC 1.11.1.13), versatile peroxidase (VP, EC1.11.1.16), and laccase (EC 1.10.3.2). LiP, MnP, and VP are hemeproteins with four or five disulfide bonds and two binding sites forstructural Ca2+ ions, and are high-redox-potential peroxidases, whichcan directly oxidize high-redox-potential substrates and/or Mn^(2±). Theperoxidase activity of MnP can be measured using the 2,6-dimethoxyphenol(2,6-DMP) oxidation assay described in Example 4, Section E, below; LiPactivity can be measured by the oxidation of veratryl alcohol toveratryl aldehyde in the presence of H₂O₂ (Orth et al., “Overproductionof lignin-degrading enzymes by an isolate of Phanerochaetechrysosporium”, Appl Environ Microbiol 1991 September; 57(9):2591-2596). Laccases are not associated with heme, but are associatedwith copper ions (usually 4 copper ions per laccase protein), and arelow-redox-potential oxidoreductases, which can only oxidizehigh-redox-potential substrates in the presence of redox mediators. Theactivity of laccases can be measured using the ABTS(2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid)) oxidationassay as described in Zhao et al., “Characterisation of a novel whitelaccase from the deuteromycete fungus Myrothecium verrucaria NF-05 andits decolourisation of dyes”, PLoS One 2012; 7(6): e38817; Epub 2012Jun. 8; one unit of activity is defined as the production of 1 micromolof product per minute.

The inducible coexpression methods of the invention can be used toproduce lignin-degrading peroxidases, which are defined as being theenzymes listed by EC number in the preceding paragraph; the BRENDA andUniProt database entries for the lignin-degrading peroxidases areincorporated by reference herein. In some embodiments, alignin-degrading peroxidase is coexpressed with a chaperone protein; infurther embodiments of the invention, a lignin-degrading peroxidase iscoexpressed with a transporter for a cofactor such as heme.

LiP can be expressed using the inducible coexpression systems of theinvention; in certain embodiments, the LiP is selected from the groupconsisting of: Phanerochaete chrysosporium (Sporotrichum pruinosum) LiPisozymes “Ligninase A” (UniProt P31837), “Ligninase B” (UniProt P31838),“Ligninase H2” (UniProt P11542), “Ligninase H8” (UniProt P06181),“Ligninase LG2” (UniProt P49012), “Ligninase LG3” (UniProt P21764),“Ligninase LG5” (UniProt P11543), and “Ligninase LG6” (UniProt P50622);Phlebia radiata “Ligninase-3” (UniProt P20010); Trametes versicolor(Coriolus versicolor) LiP isozymes “Ligninase C” (UniProt P20013), LP7(UniProt Q99057), and LP12 (UniProt Q7LHY3); and Trametopsis cervina LiP(UniProt Q3C1R8) and (UniProt Q60FD2).

In some embodiments of the invention, inducible coexpression is used toexpress MnP; in certain embodiments, the MnP is selected from the groupconsisting of: Agaricus bisporus MnP (UniProt Q5TJC2); Agrocybe praecoxMnP (UniProt G4WG41); Ganoderma lucidum MnP (UniProt COIMT8); Lenzitesgibbosa MnP (UniProt C3V8Q9); Phanerochaete chrysosporium MnP isozymesMNP1 (UniProt Q02567); H3 (UniProt P78733); and H4 (UniProt P19136);Phlebia radiata MnP isozymes MnP2 (UniProt Q70LM3) and MnP3 (UniProtQ96TS6); Phlebia sp. b19 MnP (UniProt B2BF37); Phlebia sp. MG60 MnPisozymes MnP1, MnP2, MnP3 (UniProt B1B554, B1B555, and B1B556,respectively); Pleurotus ostreatus MnP3 (UniProt B9VR21); Pleurotuspulmonarius MnP5 (UniProt Q2VT17); Spongipellis sp. FERM P-18171 MnP(UniProt Q2HWK0); and Trametes versicolor (Coriolus versicolor) MnPisozymes (UniProt Q99058, Q6B6M9, Q6B6N0, Q6B6N1, and Q6B6N2).Coexpression of MnP with protein disulfide isomerase in the presence ofheme is described in Example 4.

Laccase can be expressed using the inducible coexpression systems of theinvention; in certain embodiments, the laccase is selected from thegroup consisting of: Botryotinia fuckeliana (Botrytis cinerea) laccaseisozymes (UniProt Q12570, Q96UM2, and Q96WM9); Cerrena sp. WR1 laccaseisozymes (UniProt E7BLQ8, E7BLQ9, and E7BLRO); Cerrena unicolor(Daedalea unicolor) Laccase (UniProt B8YQ97); Ganoderma lucidum laccaseisozymes (UniProt Q6RYA2, B5G547, B5G549, B5G550, B5G551, and B5G552);Melanocarpus albomyces Laccase (UniProt Q70KY3); Pleurotus ostreatuslaccase isozymes (UniProt Q12729, Q12739, Q6RYA4, Q6RYA4, and G3FGX5);Pycnoporus cinnabarinus laccase isozymes (UniProt 059896, Q9UVQ2,D2CSG0, D2CSG1, D2CSG4, D2CSG6, and D2CSG7); Pycnoporus coccineuslaccase isozymes (UniProt D2CSF2, D2CSF5, D2CSF6, D2CSM7, and D7F484);Trametes hirsuta (Coriolus hirsutus) Laccase (UniProt Q02497); Trametesmaxima (Cerrena maxima) Laccase (UniProt DOVWU3); Trametes versicolor(Coriolus versicolor) laccase isozymes (UniProt Q12717, Q12718, andQ12719); and Trametes villosa laccase isozymes (UniProt Q99044, Q99046,Q99049, Q99055, and Q99056).

While the three main lignin-modifying enzymes of white rot fungi areLiP, MnP, and laccase, another type of peroxidase, versatile peroxidase(VP), is found in several species from the genera Pleurotus andBjerkandera. VP is of interest due to its catalytic versatility: it canoxidise LiP substrates, veratryl alcohol, methoxy-benzenes, andnon-phenolic lignin model compounds, as well as the MnP substrateMn^(2±). The versatile activity of VP can be assayed using the MnP2,6-DMP assay, or the LiP veratryl alcohol assay, or the laccase ABTSassay (see above). In some embodiments of the invention, induciblecoexpression is used to express VP; in certain embodiments, the VP isselected from the group consisting of: Bjerkandera adusta VP (UniProtA5JTV4); Pleurotus eryngii VP isozymes (UniProt 094753, Q9UR19, andQ9UVP6); and Pleurotus pulmonarius VP (UniProt I6TLM2).

Example 1

Inducible Coexpression of IgG1 Heavy and Light Chains to ProduceFull-Length Antibodies in Bacterial Cells

A. Construction of Expression Vectors

The inducible coexpression system was used to produce full-lengthantibodies, specifically mouse anti-human CD19 IgG1 antibodies, inbacterial cells. The coding sequence for the mouse anti-human CD19 IgG1heavy chain (IgG1 heavy chain′, ‘IgG1HC’, ‘heavy chain’, or ‘HC’) isprovided as SEQ ID NO:1 and is the same as that of GenBank Accession No.AJ555622.1, and specifically bases 13 through 1407 of the GenBankAJ555622.1 nucleotide sequence. The corresponding full-length mouseanti-human CD19 IgG1 heavy chain amino acid sequence is provided as SEQID NO:2 (and is the same as GenBank Accession No. CAD88275.1). Thecoding sequence for the mouse anti-human CD19 IgG1 light chain (IgG1light chain′, ‘IgG1LC’, ‘light chain’, or ‘LC’) is provided as SEQ IDNO:3 and is the same as that of GenBank Accession No. AJ555479.1, andspecifically bases 23 through 742 of the GenBank AJ555479.1 nucleotidesequence. The corresponding full-length mouse anti-human CD19 IgG1 lightchain amino acid sequence is provided as SEQ ID NO:4 (and is the same asGenBank Accession No. CAD88204.1).

Synthesis of polynucleotides encoding the IgG1 heavy and light chains,and optimization for expression in E. coli, was performed by GenScript(Piscataway, N.J.). GenScript's OptimumGene™ Gene Design system uses analgorithm, the OptimumGene™ algorithm, which takes into consideration avariety of factors involved in different stages of protein expression,such as codon usage bias, GC content, CpG dinucleotide content,predicted mRNA structure, and various cis-elements in transcription andtranslation. The optimized sequences for the IgG1 heavy and light chainsare provided as SEQ ID NO:5 and SEQ ID NO:6, respectively; theseoptimized sequences include some additional nucleotides upstream anddownstream of the coding sequence; the coding sequences are bases 25through 1419 of SEQ ID NO:5, and bases 25 through 747 of SEQ ID NO:6.The optimized coding sequence in SEQ ID NO:10 for the IgG1 light chainencodes an additional amino acid, an alanine residue, at position 2 ofthe encoded amino acid sequence, relative to the IgG1 light chain aminoacid sequence of SEQ ID NO:4. The optimized heavy and light chainsequences both contain TAA stop codons, and a preferred E. coliribosome-binding sequence (AGGAGG), located at −14 to −9 bases upstreamof the initiation codon. In addition, the IgG1 heavy chain optimizedsequence (SEQ ID NO:5) contains an NcoI restriction site comprising theATG initiation codon, and a HindIII restriction site immediatelydownstream of the TAA stop codon, while the IgG1 light chain optimizedsequence (SEQ ID NO:6) contains an NheI restriction site immediatelyupstream of the ribosome-binding sequence, and also a HindIIIrestriction immediately downstream of the TAA stop codon.

The optimized coding sequences for the IgG1 heavy and light chains wereobtained from GenScript as polynucleotide inserts cloned into pUC57. ThepBAD24 vector was obtained from the American Type Culture Collection(ATCC) as ATCC 87399, and has the nucleotide sequence shown in GenBankDatabase Accession No. X81837.1 (25 Oct. 1995); the pPRO33 vector wasobtained from the University of California (Berkeley, Calif.), and hasthe nucleotide sequence shown in SEQ ID NO:7. The nucleotide sequence ofpPRO33 was compiled from the sequences of the pBAD18 vector (GenBankAccession No. X81838.1), the E. coli genomic sequence of theprpR-P_(prpB) region, and the pBAD33 vector, as described in Guzman etal., “Tight regulation, modulation, and high-level expression by vectorscontaining the arabinose PBAD promoter”, J Bacteriol 1995 July; 177(14):4121-4130, and in U.S. Pat. No. 8,178,338 B2; May 15, 2012; Keasling,Jay. To clone the IgG1 heavy chain into the pBAD24 vector, and the IgG1light chain into the pPRO33 vector, the pUC57-HC and pUC57-LC constructswere first transformed into E. coli BL21 cells (New England Biolabs (or‘NEB’), Ipswich, Mass.) using the heat-shock method. Plasmid DNA foreach of these vectors was then obtained by a miniprep method. Unlessotherwise noted, growth of E. coli cells in liquid culture was at 37° C.with rotary shaking at 250 RPM. E. coli cell strains containing plasmids(BL21 containing pUC57-HC, BL21 containing pUC57-LC, and DH5alphacontaining pBAD24) were each grown in 8 mL LB+ampicillin (‘AMP’) medium,and E. coli DH10B cells (Life Technologies, Grand Island, N.Y.)containing pPRO33 were grown in 5 mL LB+chloramphenicol (‘CAM’) medium,for 14 hours or overnight, then cells were pelleted, lysed, and theplasmid DNA separated using a QIAprep® spin column (QIAGEN, Germantown,Md.) according to the manufacturer's protocol. For increased yields, theQIAprep® spin column protocol was performed with 100% ethanol added tothe PE buffer, and the supernatant was sent through the column twiceinstead of once.

To clone the IgG1 heavy chain into pBAD24, the purified pUC57-HC andpBAD24 plasmids were digested with NcoI and HindIII restriction enzymes(NEB), then the IgG1HC NcoI-HindIII fragment and the NcoI-HindIII-cutpBAD24 plasmid were separated from other polynucleotide fragments by gelelectrophoresis, excised from the gel, and purified using the illustraGFX Gel Band Purification Kit (GE Healthcare Life Sciences, Piscataway,N.J.) according to the manufacturer's instructions. The IgG1HCNcoI-HindIII fragment was then ligated (overnight at 16° C.) to theNcoI-HindIII-cut pBAD24 plasmid to form the pBAD24-HC expressionconstruct. Because the NcoI restriction site in the IgG1HC NcoI-HindIIIfragment comprises the ATG initiation codon of the IgG1HC codingsequence, when the IgG1HC NcoI-HindIII fragment is inserted into theNcoI-HindIII-cut pBAD24 vector, the IgG1HC coding sequence is placeddownstream of a preferred E. coli ribosome-binding sequence AGGAGGpresent in the pBAD24 vector (see FIG. 1 of Guzman et al., “Tightregulation, modulation, and high-level expression by vectors containingthe arabinose PBAD promoter”, J Bacteriol 1995 July; 177(14):4121-4130). In the resulting pBAD24-HC expression construct, theribosome-binding sequence is located at −14 to −9 bases upstream of theIgG1HC initiation codon.

To clone the IgG1 light chain into pPRO33, the purified pUC57-LC andpPRO33 plasmids were digested with NheI and HindIII restriction enzymes(NEB). Because the pPRO33 plasmid has two HindIII restriction sites,there were two fragments to be gel-purified from the pPRO33 NheI-HindIIIdigest: a 4.4 kb fragment and a 1.5 kb fragment. After gel-purificationof all the desired fragments, using an illustra GFX Gel BandPurification Kit (see above), the purified 4.4 kb pPRO33 fragment wastreated with alkaline phosphatase (also called calf intestinalphosphatase or CIP) (NEB), according to the manufacturer's instructions.The CIP-treated 4.4 kb pPRO33 fragment was purified from the phosphatasereaction mixture using an illustra GFX Purification Kit (see above), andwas then ligated to the 1.5 kb pPRO33 fragment (at 16° C. for 10.5hours). The resulting NheI-HindIII-cut pPRO33 plasmid was ligated to theIgG1LC NheI-HindIII fragment (overnight at 16° C.) to form the pPRO33-LCexpression construct. Because the NheI restriction site in the IgG1LCNheI-HindIII fragment is immediately upstream of the ribosome-bindingsequence AGGAGG in the optimized light chain sequence (SEQ ID NO:6),when the IgG1LC NheI-HindIII fragment is inserted into theNheI-HindIII-cut pPRO33 vector, the IgG1LC sequence retains itspreferred E. coli ribosome-binding sequence.

B. Inducible Coexpression of IgG1 Heavy and Light Chains in BacterialCells

The pBAD24-HC expression construct and the pPRO33-LC expressionconstruct were co-transformed into E. coli BL21 and into E. coliSHuffle® Express cells (NEB) using the heat-shock method. TransformedBL21 (pBAD24-HC/pPRO33-LC) and SHuffle® Express (pBAD24-HC/pPRO33-LC)cells were grown at 37° C. overnight in 5 mL LB broth with 30micrograms/mL CAM and 100 micrograms/mL AMP. Because the SHuffle®Express cells seem to grow more slowly than the BL21 cells, 2% (100microliters) of the overnight culture of SHuffle® Express(pBAD24-HC/pPRO33-LC), and 1% (50 microliters) of the overnight cultureof BL21 (pBAD24-HC/pPRO33-LC), were used to inoculate 5 mL of M9 minimalmedia with casamino acids and 0.2% glycerol, plus 30 micrograms/mL CAMand 100 micrograms/mL AMP. These cultures were grown until the OD₆₀₀ wasapproximately 0.5: 3.1 hours for the SHuffle® Express(pBAD24-HC/pPRO33-LC) cells and 2.5 hours for the BL21(pBAD24-HC/pPRO33-LC) cells. Prior to induction, control samples weretaken from each culture that would be grown without induction. Theremaining SHuffle® Express (pBAD24-HC/pPRO33-LC) and BL21(pBAD24-HC/pPRO33-LC) cell cultures were then induced by adding 0.2%arabinose and 50 mM propionate, and growing them in parallel with thenon-induced control samples for 6 hours. At the end of that time, allcell cultures were centrifuged to pellet the cells, and placed in a −80°C. freezer. The cell pellets were thawed on ice, and the cells werelysed using a Qproteome Bacterial Lysis Kit (QIAGEN), according to themanufacturer's instructions, with the exceptions that a Complete MiniProtease Inhibitor Cocktail Tablet (Roche, Indianapolis, Ind.) was addedto 10 mL native lysis buffer before adding the lysozyme and nuclease,and that the samples were centrifuged at 25° C. instead of at 4° C.

The soluble protein extracts from the induced cells and uninducedcontrols were separated by SDS gel electrophoresis under reducingconditions on a NuPAGE® 4-12% Bis-Tris gel (Life Technologies, GrandIsland, N.Y.). The gel was stained using RAPIDstain™ (G-Biosciences, St.Louis, Mo.). As shown in FIG. 3, a protein band (heavy chain) is seenmigrating at 51 kDa and another protein band (light chain) at 26 kDa;these bands are present in the induced cells but not in the uninducedcells. The same soluble protein extracts from induced and uninducedSHuffle® Express and BL21 cells containing both the pBAD24-HC andpPRO33-LC inducible expression vectors were separated by gelelectrophoresis under native (non-reducing) conditions on a Novex®10-20% Tris-Glycine gel (Life Technologies). The gel was stained usingRAPIDstain™ (G-Biosciences). As shown in FIG. 4, a protein band (IgG1antibody comprising heavy and light chains) migrates at 154 kDa; thisband is present in the induced SHuffle® Express cells, but issignificantly reduced or absent in the induced BL21 cells and in theuninduced cells.

Example 2

Characterization of Expression Constructs and IgG1 Full-LengthAntibodies Produced in Bacterial Cells

A. Characterization of Expression Vectors

The sequence of the pBAD24-HC and pPRO33-LC expression constructs isconfirmed using the primers shown in the following table to initiatedideoxy chain-termination sequencing reactions, along with other primersdesigned as needed. The nucleotide sequence of the prpBCDE promoter andthe region of the pPRO24 vector upstream of the MCS, as shown in FIG. 1Cof U.S. Pat. No. 8,178,338 B2, is used to design at least one forwardoligonucleotide primer to sequence coding sequences cloned into the MCSof pPRO expression vectors.

TABLE 4 Oligonucleotide primers SEQ ID Primer NO: Sequence CommentsIgG1HC Fc  8 5′-TTC ACC ATG GAA Matches bases 780-807 of SEQ ID forwardGTT TCA TCG GTC TTT NO: 5, adds a NcoI site at 5' end ATT TTC CCG-3′IgG1HC Fc  9 5′-AGC CAA GCT TTT Match in reverse orientation to basesreverse ATT TAC CCG GCG AGT 1398-1429 of SEQ ID NO: 6 GGG AC-3′pBAD24/33 10 5′-CTG TTT CTC CAT Located between the pBAD promoterforward ACC CGT T-3′ and the MCS in all pBAD vectors pBAD24/33 115′-CTC ATC CGC CAA Just downstream of MCS in pBAD and reverse #1AAC AG-3′ pPRO vectors, separated from MCS HindIII site by 2 bases (GG)pBAD24/33 12 5′-GGC TGA AAA TCT Downstream of reverse primer #1 inreverse #2 TCT CT-3′ pBAD and pPRO vectors and overlapswith it, last two bases are first two of #1

B. Detection of Mouse IgG1 Full-Length Antibodies on a Western Blot

Protein gels used to separate soluble protein extracts byelectrophoresis (NuPAGE® 4-12% Bis-Tris gels or Novex® 10-20%Tris-Glycine gels), as described in Example 1, are placed into a XCellII™ Blot Module (Life Technologies, Grand Island, N.Y.). Current isapplied to the gel in accordance with the manufacturer's instructions,resulting in the transfer of proteins from the gel to a nitrocellulosemembrane. The nitrocellulose membrane is then incubated with a primaryantibody, anti-mouse IgG, at 4° C. overnight. The nitrocellulosemembrane is then washed to remove unbound antibody, and incubated with asecondary antibody, goat anti-mouse IgG conjugated to alkalinephosphatase, for one hour at room temperature. The nitrocellulosemembrane is then washed to remove unbound secondary antibody, incubatedwith a solution containing nitroblue tetrazolium (NBT) and5-bromo-4-chloro-indolyl-phosphate (BCIP) to stain the protein band(s),and then washed to remove excess staining solution.

Example 3

Introduction of Genomic Alterations into Host Cells to FacilitateCoexpression

As described above, certain changes in host cell gene expression canimprove the coexpression of the desired gene product(s). The followingdeletions and alterations were made in the E. coli SHuffle® Express hostcell genome by Gene Bridges GmbH (Heidelberg, Germany) using arecombineering method, described as deletion by counterselection, thatseamlessly removes genomic sequences. A deletion of the host cell araBADoperon was made to reduce arabinose catabolism by the host cell, so thatmore of the arabinose inducer will be available for induction of acoexpressed gene product from an expression construct comprising thearaBAD promoter. This deletion removes 4269 basepairs of the araBADoperon, corresponding to position 70,135 through 65,867 (minus strand)of the E. coli genome (positions within genomic nucleotide sequences areall given as in Table 1), so that most of the native araBAD promoterthrough all but a few codons of the AraD coding region are removed. Thenucleotide sequence (minus strand) around the deletion junction(position 70,136|position 65,866) is: TTAT|TACG. Another deletion wasmade within the sbm-ygfDGH (also called scpA-argK-scpBC) operon,eliminating the function of genes involved in the biosynthesis of2-methylcitrate, to increase sensitivity of the host cell'spropionate-inducible promoter to exogenously supplied propionate. Thesbm-ygIDGH deletion removes 5542 basepairs (position 3,058,754 through3,064,295 of the E. coli genome), taking out the sbm-ygfDGH promoter andall of the operon except for the last codon of the ygfH coding sequence,while leaving the adjacent ygfI coding sequence and stop codon intact.The nucleotide sequence (plus strand) around the deletion junction(position 3,058,753|position 3,064,296) is: ACAA|GGGT. In addition tothese deletions made in the E. coli SHuffle® Express host cell genome,Gene Bridges GmbH introduced a point mutation in the genomic rpsL genecoding sequence, which extends on the minus strand from position3,472,574 through 3,472,200, changing the A at position 3,472,447 to aG, altering the codon for Lys43 to a codon for Arg, which results in astreptomycin-resistant phenotype when the mutant rpsL-Arg43 gene isexpressed. Another alteration to the host cell genome, allowing for moretightly controlled inducible expression as described above, is to makethe araE promoter constitutive rather than responsive to arabinose. Mostof the native araE promoter, including CRP-cAMP and AraC binding sites,was removed by deleting 97 basepairs (position 2,980,335 through2,980,239 (minus strand)) and replacing that sequence with the35-basepair sequence of the constitutive J23104 promoter, with theresulting junction site sequences: TGAA|TTGA←J23104 promoter→TAGC|TTCA.An E. coli host cell, such as an E. coli SHuffle® Express host cell,with any of these genomic alterations, or any combination of them, canbe employed in the inducible coexpression of gene products.

Example 4

Inducible Coexpression of Manganese Peroxidase and Protein DisulfideIsomerase in the Presence of Heme

A. Construction of Expression Vectors

Manganese peroxidase (′MnP′; also called manganese-dependant peroxidase)is an enzyme that enables some types of fungi to degrade lignin tocarbon dioxide, and to mediate oxidation of a wide variety of organicpollutants. One example of manganese peroxidase is the H4 isozyme(referred to herein as ‘MnP-H4’) of the white rot fungus Phanerochaetechrysosporium (UniProtKB/Swiss-Prot Accession No. P19136). In itsfunctional form, MnP-H4 is associated with a manganese ion (Mn2⁺), aniron-containing heme molecule, and two calcium ions (Can; MnP-H4 alsohas five disulfide bonds (Sundaramoorthy et al., “The crystal structureof manganese peroxidase from Phanerochaete chrysosporium at 2.06-Åresolution”, J Biol Chem 1994 Dec. 30; 269(52): 32759-32767). Thermalinactivation of the MnP-H4 enzyme involves a loss of the interactionsbetween MnP-H4 and the calcium ions. Creating an additional disulfidebond in MnP-H4 by altering the amino acid sequence of MnP-H4 tosubstitute cysteine for another amino acid at two positions near thedistal calcium interaction site, such as an MnP-H4 A48C/A63C variant,makes the resulting MnP-H4 A48C/A63C enzyme more resistant to thermalinactivation (Reading and Aust, “Engineering a disulfide bond inrecombinant manganese peroxidase results in increased thermostability”,Biotechnol Prog 2000 May-June; 16(3): 326-333). To express MnP-H4 in away that promotes formation of the disulfide bonds, an expressionconstruct was created encoding an MnP-H4 enzyme lacking the signalpeptide, so that the protein remains in the oxidizing cytoplasmicenvironment of the host cell (E. coli SHuffle® Express).

The amino acid sequence of MnP-H4 without a signal peptide is shown asSEQ ID NO:13; this form of the MnP-H4 protein has an initial methionineresidue attached to the predicted mature amino acid sequence, startingat A14 of the full-length amino acid sequence, as described in Pease etal., “Manganese-dependent peroxidase from Phanerochaete chrysosporium.Primary structure deduced from cDNA sequence”, J Biol Chem 1989 Aug. 15;264(23): 13531-13535. In other references, such as Sundaramoorthy etal., “The crystal structure of manganese peroxidase from Phanerochaetechrysosporium at 2.06-Å resolution”, J Biol Chem 1994 Dec. 30; 269(52):32759-32767, the mature MnP-H4 amino acid sequence is indicated asstarting at A25 of the full-length amino acid sequence. Proteinscomprising amino acids 13 though 370 of SEQ ID NO:13 (which correspondsto the shorter mature amino acid sequence) are also produced by themethods of the invention, and in certain embodiments have an initialmethionine residue attached to amino acids 13 though 370 of SEQ IDNO:13, and thus have the amino acid sequence shown as SEQ ID NO:23. Thenucleotide sequence that has been optimized for the expression of theSEQ ID NO:13 form of MnP-H4 in E. coli is shown as SEQ ID NO:14;expression constructs comprising SEQ ID NO:14 were used in the methodsof the invention to express MnP-H4. Nucleotides 37 through 1110 of SEQID NO:14 encode the shorter MnP-H4 mature amino acid sequence (startingat A25 of the full-length amino acid sequence); polynucleotidescomprising nucleotides 37 through 1110 of SEQ ID NO:14 are used in someembodiments of the invention for production of MnP-H4 protein, and incertain embodiments comprise an ATG codon for an initial methionineresidue attached to the 5′ end of the nucleotide sequence of 37 through1110 of SEQ ID NO:14.

A similar expression construct is created for expression of an MnP-H4protein corresponding to the A48C/A63C MnP-H4 protein and lacking asignal peptide (amino acid sequence shown as SEQ ID NO:15; optimizedcoding sequence shown as SEQ ID NO:16). Due to the additional twelveamino acids in SEQ ID NO:15 as compared to the shorter mature MnP-H4amino acid sequence, the alanine-to-cysteine alterations are indicatedas A60C/A75C in SEQ ID NO:15. Additional examples of expressionconstructs that are used in the methods of the invention encode aprotein comprising SEQ ID NO:15, or amino acids 13 though 370 of SEQ IDNO:15, or an initial methionine residue attached to amino acids 13though 370 of SEQ ID:15. In certain embodiments, expression constructsthat are used in the methods of the invention comprise SEQ ID NO:16, ornucleotides 37 through 1110 of SEQ ID:16, or an ATG codon for an initialmethionine residue attached to the 5′ end of the nucleotide sequence of37 through 1110 of SEQ ID:16. Another variation of the MnP-H4 amino acidsequence occurs at position 105 of the full-length amino acid sequence,where a serine is changed to an asperagine. The methods of the inventionare used to produce MnP-H4 proteins with this variation (serine changedto asperagine at position 93 of SEQ ID NO:13 or SEQ ID NO:15), withexpression constructs comprising nucleotide sequences having a G atposition 278 of SEQ ID NO:14 or SEQ ID NO:16 changed to an A, alteringthe AGC codon for serine to an AAC codon for asperagine.

In addition to promoting the formation of disulfide bonds in MnP-H4, toproduce fully active MnP-H4 enzyme, the enzyme is optimally expressed inthe presence of heme. To allow the E. coli host cell to take upheme-containing molecules such as hemin from the medium, a nucleotidesequence encoding the E. coli O157:H7 ChuA outer-membrane hemin-specificreceptor is also included in the MnP-H4 expression construct. The ChuApolypeptide has the same N-terminal amino acid sequence, including thesignal peptide, as the native E. coli O157:H7 str. EC4113 ChuA protein(SEQ ID NO:17), so that it will be inserted into the outer membrane ofthe E. coli host cell. The optimized coding sequence for the ChuApolypeptide (SEQ ID NO:17) is shown at positions 68 through 2047 of SEQID NO:19. The ChuA amino acid sequence shown in SEQ ID NO:17, from E.coli O157:H7 str. EC4113, differs from that of the ChuA amino acidsequence from E. coli CFT073 (NCBI Gene ID No. 1037196) by having avaline (EC4113) instead of an isoleucine (CFT073) at position 106 of SEQID NO:17. A V1061 change in the ChuA amino acid sequence can be encodedby a change in the GTG Val codon to an ATT or ATC Ile codon at positions383-385 of SEQ ID NO:19. Other variations in the amino acid sequence ofChuA proteins used in the expression of heme-associated proteinsinclude, for example, a change from glutamic acid to glycine at position259 of SEQ ID NO:17 (encoded by a change in the GAG Glu codon to a GGTor GGC Gly codon at positions 842-844 of SEQ ID NO:19), or a change fromglutamic acid to asparagine at position 262 of SEQ ID NO:17 (encoded bya change in the GAG Glu codon to a GAT or GAC Asp codon at positions851-853 of SEQ ID NO:19).

Optimization for expression in E. coli and synthesis of polynucleotidescorresponding to SEQ ID NOs 18, 19, and 22 was performed by DNA2.0(Menlo Park, Calif.). SEQ ID NO:18 encodes the MnP-H4 protein, and isdesigned to be inserted immediately downstream of a promoter, such as aninducible promoter. The SEQ ID NO:18 nucleotide sequence starts at its5′ end with a GCTAGC NheI restriction site, and has an AGGAGG ribosomebinding site at nucleotides 7 through 12 of SEQ ID NO:18, followed bythe optimized MnP-H4 coding sequence at nucleotides 21 through 1130 ofSEQ ID NO:18. Downstream of the MnP-H4 stop codon is the B0015 doubleterminator, from position 1142 through 1270 of SEQ ID NO:18, followed bya TCTAGA XbaI restriction site. The nucleotide sequence of the B0015double terminator was obtained from the partsregistry.org website. SEQID NO:19 encodes ChuA, and includes a constitutive promoter, so thisexpression construct for ChuA could be placed in any expression vectoror within the host cell genome; because the ChuA coding sequence hasbeen placed under the control of the J23104 constitutive promoter, itstranscription is no longer subject to repression by Fur. In thisembodiment, SEQ ID NO:19 starts at its 5′ end with a TCTAGA XbaIrestriction site, and is designed to be placed within an expressionvector 3′ to the sequences of SEQ ID NO:18. Nucleotides 7 through 41 ofSEQ ID NO:19 are the J23104 constitutive promoter and nucleotides 50through 61 of SEQ ID NO:19 are the B0034 ribosome binding site, bothplaced upstream of the ChuA coding sequence at nucleotides 68 through2047 of SEQ ID NO:19; the nucleotide sequences of J23104 and B0034 wereobtained from the partsregistry.org website. SEQ ID NO:19 ends with aGTCGAC SalI restriction site. The XbaI sites in SEQ ID NO:18 and SEQ IDNO:19 allow these nucleotide sequences to be ligated together at a XbaIsite; the nucleotide sequence of the resulting MnP-H4 ChuA expressionconstruct is shown in SEQ ID NO:20. The NheI and SalI sites in SEQ IDNOs 18 and 19, respectively, and in SEQ ID NO:20, allow the MnP-H4 ChuAexpression construct to be inserted into an expression vector such aspPRO33 using the NheI and SalI restriction sites in its multiple cloningsite.

To facilitate production of correctly folded MnP-H4 enzyme, MnP-H4 wascoexpressed with the chaperone protein disulfide isomerase (‘PDI’) fromHumicola insolens, a thermophilic, cellulolytic, and saprophytic soilhyphomycete (soft-rot fungus). The amino acid sequence of PDI that wascoexpressed is shown as SEQ ID NO:21; it lacks the signal peptide of thenative protein so that it remains in the host cell cytoplasm as theMnP-H4 polypeptides are produced. The nucleotide sequence encoding PDIwas also optimized for expression in E. coli; the expression constructfor PDI is shown as SEQ ID NO:22. SEQ ID NO:22 contains a GCTAGC NheIrestriction site at its 5′ end, an AGGAGG ribosome binding site atnucleotides 7 through 12, the PDI coding sequence at nucleotides 21through 1478, and a GTCGAC SalI restriction site at its 3′ end. Thenucleotide sequence of SEQ ID NO:22 was designed to be insertedimmediately downstream of a promoter, such as an inducible promoter. TheNheI and SalI restriction sites in SEQ ID NO:22 were used to insert itinto the multiple cloning site of the pBAD24 expression vector.

The synthesized expression constructs comprising SEQ ID NO:20 and SEQ IDNO:22, and the pPRO33 and pBAD24 vectors, were cut with NheI and SalIrestriction enzymes, and the synthesized expression construct fragmentswere ligated into the vectors to create pPRO33-MnP-ChuA and pBAD24-PDI,as described immediately above and in Example 1. E. coli SHuffle®Express cells (New England Biolabs (or ‘NEB’), Ipswich, Mass.) wereco-transformed with the resulting expression vectors (pPRO33-MnP-ChuAand pBAD24-PDI) using the heat-shock method.

B. Inducible Coexpression of Manganese Peroxidase and Protein DisulfideIsomerase in Bacterial Cells

Host cells co-transformed with the pPRO33-MnP-ChuA and pBAD24-PDIexpression vectors (SHuffle® Express(pPRO33-MnP-ChuA/pBAD24-PDI) cells)were used to inoculate four shake tubes each containing 5 ml LB brothplus 34 micrograms/mL CAM and 100 micrograms/mL AMP. After incubation(at 30° C. with rotary shaking at 250 RPM) for 16 hours, the cells werespun at 4000 rpm for 10 minutes at 4° C., the LB broth decanted off, andthe cells were resuspended in 4×400 microliters of M9 minimal media withcasamino acids and 0.2% glycerol, plus 34 micrograms/mL CAM and 100micrograms/mL AMP (‘M9-CA-gly+CAM+AMP’). Then 75 microliters of thecombined volume of 1.6 ml of resuspended cells was added to each of tenshake tubes containing 5 ml M9-CA-gly+CAM+AMP, and the OD₆₀₀ of theresulting culture was determined to be 0.6. Hemin (Sigma-Aldrich, St.Louis, Mo.) was added to a final concentration of 8 micromolar in alltubes except tube 1, the uninduced control. The L-arabinose andpropionate inducers were added to tubes 2-10 in the followingconcentrations:

Tube 1: Not Induced (no hemin, no propionate, no arabinose) Tube 2: 50mM propionate 0.002% arabinose Tube 3: 25 mM propionate 0.002% arabinoseTube 4: 12.5 mM propionate 0.002% arabinose Tube 5: 50 mM propionate0.01% arabinose Tube 6: 25 mM propionate 0.01% arabinose Tube 7: 12.5 mMpropionate 0.01% arabinose Tube 8: 50 mM propionate 0.05% arabinose Tube9: 25 mM propionate 0.05% arabinose Tube 10: 12.5 mM propionate 0.05%arabinose

The cells were induced at 25° C. for 12 hours with rotary shaking, thenspun down and placed in a −80° C. freezer. The cell pellets were thawedon ice, and the cells were lysed using a Qproteome Bacterial Lysis Kit(QIAGEN), according to the manufacturer's instructions; ProteaseInhibitor Cocktail was not added, and the samples were centrifuged at 4°C. The soluble protein extracts from the induced cells and uninducedcontrol were separated by SDS gel electrophoresis under reducingconditions on a 10% Bis-Tris gel (Life Technologies, Grand Island,N.Y.). The gel was stained using RAPIDstain™ (G-Biosciences). As shownin FIG. 5, lysate from induced cells contained proteins corresponding toPDI (53 kDa) and MnP-H4 (39 kDa), indicating that these proteins wereexpressed as soluble proteins in the bacterial cells. The greatestamount of soluble MnP-H4 was produced by induction with 50 mM propionateand 0.002% arabinose (lane 2).

C. Inducible Coexpression of an Alternate Mature Form of ManganesePeroxidase Along with Protein Disulfide Isomerase, and Measurement ofMnP-H4 Activity

Expression vectors were prepared to express a more fully truncatedversion of MnP-H4, referred to as MnP-H4 FT, which corresponds to themature version of MnP-H4 protein as described in Sundaramoorthy et al.,1994 (cited above). The MnP-H4_FT amino acid sequence thus has aninitial methionine residue attached to amino acids 13 though 370 of SEQID NO:13, and is provided as SEQ ID NO:23. In this experiment, theMnP-H4_FT coding sequence optimized for expression in E. coli (derivedfrom the optimized MnP-H4 coding sequence described above), and the ChuAcoding sequence, were expressed in the pBAD24 vector, and the PDI codingsequence (similarly optimized for expression in E. coli as describedabove) was expressed in the pPRO33 vector. The pBAD24-MnP_FT-ChuAexpression construct was prepared by PCR-amplifying the MnP-H4 codingsequence and terminator from a template comprising the nucleotidesequence of SEQ ID NO: 18, using the forward and reverse primers of SEQID NOs 24 and 25, respectively. Use of the SEQ ID NO:24 forward primerplaces a NcoI restriction site and an ATG codon immediately upstream ofthe coding seuqence for amino acids 13 though 370 of SEQ ID NO:13, andthus creates a coding sequence for the MnP-H4_FT amino acid sequence ofSEQ ID NO:23. The ChuA expression construct sequences were PCR-amplifiedfrom a template comprising the nucleotide sequence of SEQ ID NO:19,using the forward and reverse primers of SEQ ID NOs 26 and 27,respectively. The MnP-H4_FT and ChuA PCR products were cut with NcoI andSalI, and with SalI and HindIII, respectively, and were gel-purified andligated together into the pBAD24 vector, which had been cut with NcoIand HindIII, CIP-treated, and gel-purified. The ligatedpBAD24-MnP_FT-ChuA products comprise the MnP-H4_FT coding sequenceexpressed from the pBAD promoter, followed by the B0015 doubleterminator, the J23104 constitutive promoter, the B0034 ribosome bindingsite, and the ChuA protein-coding sequence. This pBAD24-MnP_FT-ChuAexpression construct has the nucleotide sequence provided in SEQ IDNO:28.

The pPRO33-PDI expression construct was made by cutting the pPRO33vector and the PDI expression construct, optimized for expression in E.coli as described above and comprising the nucleotide sequence of SEQ IDNO:22, with NheI and SalI, treating the cut pPRO33 vector with CIP,gel-purifying the fragments, and ligating them together. The resultingpPRO33-PDI expression construct has the nucleotide sequence provided inSEQ ID NO:29. The pBAD24-MnP_FT-ChuA and pPRO33-PDI expressionconstructs were used to cotransform E. coli SHuffle® Express cells (NEB)at 37 degrees C. overnight, to form SHuffle®Express(pBAD24-MnP_FT-ChuA/pPRO33-PDI) cells.

SHuffle® Express(pBAD24-MnP_FT-ChuA/pPRO33-PDI) and control SHuffle®Express(pBAD24/pPRO33) cells were used to inoculate 5 milliliters of LBmedia+CAM (34 micrograms/milliliter)+AMP (100 micrograms/milliliter) andgrown overnight at 30 degrees C. with shaking at 250 rpm. Cells werespun down at 4000 rpm for 10 minutes at 4 degrees C., and resuspended,first in 400 microliters of M9 minimal (CA_noGlycerol: with casaminoacids as a carbon source, but no glycerol)+CAM+AMP media, and then 75microliters of that was added to 5 milliliters of M9 (CA_noGlycerol)minimal media+CAM+AMP. After the culture, grown at 30 degrees C. withshaking at 250 rpm, reached an OD600 of 0.6, ten aliquots of SHuffle®Express(pBAD24-MnP_FT-ChuA/pPRO33-PDI) cells were taken and induced invarying concentrations of hemin and of the arabinose and propionateinducers. Sample 1, the control, received no hemin and no inducers; theother nine samples had hemin added to a final concentration of 8micromolar; arabinose concentrations of 0.025%, 0.05%, or 0.1%; andpropionate concentrations 12.5 mM, 25 mM, or 50 mM. The control SHuffle®Express(pBAD24/pPRO33) cells (no inserts) were also included in theinduction process, with an ‘induced’ sample and an uninduced controlsample. The cells were induced for 12 hours at 25 degrees C. withshaking at 250 rpm, then were spun down and stored at −80 degrees C. Tovisualize the proteins produced by the induced coexpression, the frozencell pellets were thawed and lysed using a Qproteome Bacterial Lysis Kit(QIAGEN) according to the instructions, but with 35 microliters of lysisbuffer used for each 1 microliter of bacterial culture. Soluble proteinextracts from the SHuffle® Express(pBAD24-MnP_FT-ChuA/pPRO33-PDI)induced cells and the uninduced control were separated by SDS gelelectrophoresis under reducing conditions on a 10% Bis-Tris gel; sampleswere heated at 70 degrees C. for 10 minutes prior to loading on the gel.After staining the gel with RAPIDstain™ (gel not shown), there werevisible bands corresponding to MnP-H4_FT and PDI in all the inducedsamples but not in the uninduced control, and the apparent density ofbands on the gel indiated that the combination of 0.1% arabinose and 50mM propionate produced the most MnP-H4_FT protein, and that higherarabinose concentrations generally produced more MnP-H4_FT, while lowerarabinose concentrations generally produced more PDI, possibly due tocatabolite repression of the propionate promoter by arabinose.

To determine the activity levels of the MnP-H4_FT produced by theinducible coexpression, 0.5 milliliters of the soluble cell lysisfraction, derived from the coexpression of MnP-H4_FT protein at 0.1%arabinose and 50 mM propionate, was dialyzed against 10 millimolarsodium acetate, 5 millimolar CaCl₂, ph 4.5, using a 0.5-milliliter20,000-MWCO (molecular weight cutoff) Slide-A-Lyzer™ (Thermo FisherScientific Inc., Waltham Mass.). The sample was dialyzed at 4 degrees C.for 2 hours, the buffer changed for fresh buffer, and dialysis continuedat 4 degrees C. overnight. There was protein precipitation at the end ofthe dialysis, but the MnP-H4_FT protein was still soluble and active, asshown by gel electrophoresis, and by a colorimetric enzymatic activityassay. The dialyzed protein sample containing MnP-H4_FT, along with thesoluble protein fractions obtained from the SHuffle®Express(pBAD24-MnP_FT-ChuA/pPRO33-PDI) uninduced control cell, and fromthe no-insert SHuffle® Express(pBAD24/pPRO33) induced and uninducedcontrol cells, were separated by SDS gel electrophoresis under reducingconditions on a 10% Bis-Tris gel; samples were heated at 70 degrees C.for 10 minutes prior to loading on the gel. After staining the gel withRAPIDstain™, as shown in FIG. 6, bands corresponding to PDI (54 kDa) andMnP-H4_FT (38 kDa) are clearly visible in the dialyzed protein sampleproduced by coexpression in 0.1% arabinose and 50 mM propionate (FIG. 6,Lane 2), and not visible in any of the control samples (Lane 3—SHuffle®Express(pBAD24-MnP_FT-ChuA/pPRO33-PDI) uninduced control; Lane4—no-insert induced control; Lane 5—no-insert uninduced control). Theenzymatic activity of the MnP-H4_FT protein produced by the induciblecoexpression was assayed using the colorimetric manganese peroxidaseassay described in Example 4 section E below, and the coexpressedMnP-H4_FT protein was shown to have comparable manganese peroxidaseactivity to that of a positive control MnP sample.

D. Production and Purification of Manganese Peroxidase

Host cells that have been co-transformed with MnP-ChuA and PDIexpression vectors are streaked onto LB plates containingchloramphenicol and ampicillin. Single colonies are picked and each isused to inoculate a shake tube containing 15 ml LB+CAM+AMP broth. Afterincubation (at 30-37° C. with rotary shaking at 250 RPM) for an adequatelength of time to generate stationary phase cultures, 5 ml from eachtube with successful growth is used to inoculate an Erlenmeyer shakeflask containing 100 ml LB+CAM+AMP broth. After further incubation (at30-37° C. with rotary shaking at 250 RPM) sufficient to generatestationary phase cultures, a sample from each shake flask is checked foradequate cell density. An appropriate volume from a shake flask isintroduced into a sterilized and pH-calibrated bioreactor and grown at30-37° C. with agitation; after a period of growth, the cells are grownin medium containing hemin or another source of heme, until the cellsreach an OD₆₀₀ of approximately 0.5. The cells are then induced byadding arabinose and propionate; for example, 0.02% arabinose and 50 mMpropionate, or as determined by the titration methods of Example 7, andgrowth at 25-30° C. with agitation is continued. After incubation thecells are recovered from the growth medium, lysed, and the lysissupernatant (soluble protein extract) is collected. The MnP-H4 proteinis purified from the soluble protein extract using methods such as fastprotein liquid chromatography (FPLC) with a size-exclusion column or anion-exchange column.

E. Assay for Manganese Peroxidase Activity

The amount of manganese peroxidase activity in a sample, and thus theconcentration of active manganese peroxidase, can be determined bytesting it for the ability to oxidize 2,6-dimethoxyphenol (2,6-DMP) tocoerulignone in the presence of manganese and hydrogen peroxide. Thefollowing assay is for a 10-microliter sample; alternate amounts for a1-microliter sample are given in parentheses. The following is added toa spectrophotometer cuvette before sample addition: 0.590 ml dH₂O (0.599ml dH₂O); 0.1 ml malonate disodium salt monohydrate (MDSH) solution (5 gMDSH in 60 ml dH₂O, pH 4.5); and 0.1 ml MnSO₄.H₂O solution (0.06 gMnSO₄.H₂O in 90 ml dH₂O) Immediately before measuring, 0.1 ml 2,6-DMPsolution (0.014 g 2,6-dimethoxyphenol in 90 ml dH₂O) and the 10microliter (1 microliter) sample is added to the cuvette. The cuvette isplaced in the spectrophotometer and zeroed at 469 nm, and 0.1 ml freshH₂O₂ (3.4 microliters 30% H₂O₂ in 30 ml dH₂O) is then added to thecuvette. The cuvette contents are mixed by pipetting up and down threetimes. One minute after addition of H₂O₂, the OD at 469 nm is measured.If the OD is greater than 0.2, the sample size is decreased to 1microliter, or the sample to be measured is diluted; if the OD is lessthan 0.005, the sample size is increased to 0.1 ml and the dH₂O volumeis decreased to 0.500 ml, with all other volumes remaining the same; theassay is then repeated.

The measured absorbance is used to calculate the enzyme concentration(EC) in Units/L according to the following equation:

${{EC}\left\lbrack {U\text{/}L} \right\rbrack} = \frac{({Absorbance})\left( {{Assay}\mspace{14mu} {{volume}\mspace{14mu}\lbrack{ml}\rbrack}} \right)\left( {10^{6}\mspace{14mu} {\mu mol}\text{/}{mol}} \right)\left( {1\mspace{14mu} {cm}} \right)}{\left( ɛ_{1\mspace{14mu} c\; m} \right)*\left( {{Sample}\mspace{14mu} {{volume}\mspace{14mu}\lbrack{ml}\rbrack}} \right)}$

where ε_(1 cm)=49600 absorbance/M·cm·min and path length=1 cm. Theenzyme concentration as calculated above is converted to mg/L accordingto the following equation: EC [mg/L]=enzyme concentration [U/L]/enzymespecific activity [U/mg], where a standard enzyme specific activity forMnP is 160 U/mg. The enzyme specific activity is calculated for any MnPsample by independently measuring the concentration of protein in thesample in mg/L, for example by spectrophotometric analysis at 280 nm.When the enzyme concentration (EC) is determined using the above 2,6-DMPoxidation assay, the specific activity in U/mg is calculated as: EC[U/L]/concentration [mg/L]=specific activity [U/mg].

Example 5

Inducible Coexpression of Infliximab

Infliximab is a chimeric monoclonal antibody that binds to TNF-alpha, aninflammatory cytokine, and is used in the treatment of conditions thatinvolve TNF-alpha such as autoimmune diseases (Crohn's disease,rheumatoid arthritis, psoriasis, etc.). Infliximab is formed from aheavy chain (amino acid sequence shown as SEQ ID NO:30) and a lightchain (amino acid sequence shown as SEQ ID NO:31); each of these chainshas a variable domain sequence derived from mouse anti-TNF-alphaantibodies, and a human constant domain. Codon optimization forexpression in E. coli and synthesis of polynucleotides encoding SEQ IDNOs 30 and 31 was performed by DNA2.0 (Menlo Park, Calif.). Theexpression construct formed by ligating the optimized coding sequencefor the infliximab heavy chain into the multiple cloning site (MCS) ofthe pBAD24 expression vector is pBAD24-Infliximab_HC, which has thenucleotide sequence shown as SEQ ID NO:32. The expression constructformed by ligating the optimized coding sequence for the infliximablight chain into the MCS of the pPRO33 expression vector ispPRO33-Infliximab_LC, which has the nucleotide sequence shown as SEQ IDNO:33. The pBAD24-Infliximab_HC and pPRO33-Infliximab_LC expressionconstructs are used to transform E. coli SHuffle® Express cells (NEB) at37 degrees C. overnight, creating SHuffle®Express(pBAD24-Infliximab_HC/pPRO33-Infliximab_LC) cells. These cellsare grown generally as described in Examples 1 and 4, including theaddition of selective compounds such as ampicillin and/orchloramphenicol as needed, with the exception that iron is preferablybut optionally added to the LB growth medium, for example in the form ofFeSO₄.7H₂O at a concentration of 3.0 miligrams per liter. Cells are spundown and resuspended in M9 medium, preferably but optionally withacetate (for example, 0.27% sodium acetate (C₂H₃O₂Na)) added as thecarbon source along with casamino acids (which provide essential aminoacids, along with being a carbon source), and also preferably butoptionally with iron supplementation of the media as described above,and grown until the optical density (OD600) of the culture reaches 0.8.(See Paliy and Gunasekera, “Growth of E. coli BL21 in minimal media withdifferent gluconeogenic carbon sources and salt contents”, ApplMicrobiol Biotechnol 2007 January; 73(5): 1169-1172; Epub 2006 Aug. 30;Erratum in: Appl Microbiol Biotechnol 2006 December; 73(4): 968). Atthat point cells are induced by addition of arabinose (initially atconcentrations including 0.1%) and propionate (initially atconcentrations including 50 mM). Adjustment of the concentrations ofarabinose and propionate can be made as described in EXAMPLE 7, and theinfliximab antibodies produced are purified and characterized asdescribed in EXAMPLE 9 through EXAMPLE 11.

Example 6

Inducible Coexpression of Proteins in Yeast Cells

Expression constructs for the inducible coexpression in yeast host cellsof MnP-H4_FT with PDI, and of the mouse anti-human CD19 IgG1 antibodyheavy and light chains, were created by the following method carried outby GenWay Biotech Inc. (San Diego Calif.). The pJ1231-03C and pJ1234-03Cvectors (DNA2.0, Menlo Park, Calif.) were used as the backbone part ofthe yeast expression constructs, as they contain elements necessary forplasmid maintenance in either E. coli (pUC origin of replication) orSaccharomyces cerevisiae (2-micron circle origin of replication, alongwith selectable markers useful in either host: KanR (E. coli) and Leu2(yeast) in pJ1231-03C; AmpR (E. coli) and Ura3 (yeast) in pJ1234-03C.The nucleotide sequences of the pJ1231-03C and pJ1234-03C vectors areshown as SEQ ID NOs 34 and 35, respectively. The pJ1231-03C andpJ1234-03C vectors were treated with BfuA1 restriction endonuclease(NEB, Catalog No. R0701S); fragments of the vectors that were notretained after BfuA1 digestion comprise an expression promoter, theDasherGFP coding sequence, and the CYC1 terminator sequence. PCR wasperformed on four expression constructs containing coding sequences forthe polypeptides to be expressed, to create sequences that were thenligated into the BfuA1-cut pJ1231-03C and pJ1234-03C vectors. All PCRreactions were performed using Platinum® Pfx DNA Polymerase (LifeTechnologies, Grand Island, N.Y., Catalog No. 11708021), and the QIAEXII DNA purification kit (QIAGEN, Catalog No. 20051) was used forpurification of PCR-product and vector fragments, according to themanufacturers' instructions.

The pBAD24-MnP_FT-ChuA expression construct (SEQ ID NO:28), containingthe AraC coding sequence, the pBAD arabinose-inducible promoter, and theMnP-H4_FT and ChuA coding sequences, is used as the template in twoseparate PCR reactions in order to add a 6×His tag at the C-terminalends of the MnP-H4_FT coding sequence and the ChuA coding sequence, andBsaI restriction sites for cloning into the vectors. (Preferably, butoptionally, the ChuA coding sequence is not altered to add a His tag atits C-terminal end; this can be accomplished by using a primer similarin sequence to that of SEQ ID NO:39, but without bases 21 through 38 ofSEQ ID NO:39.) The primers used in these two PCR reactions are (1) theBsaI-AraC-MnP-H4_FT primer (SEQ ID NO:36) and theMnP-H4_FT-6×His-reverse primer (SEQ ID NO:37), and (2) theMnP-H4_FT-6×His-forward primer (SEQ ID NO:38) and the ChuA-6×His-BsaIprimer (SEQ ID NO:39). A further PCR reaction is then performed on thetwo purified PCR products, using the BsaI-AraC-MnP-H4_FT andChuA-6×His-BsaI primers, to create a single product(AraC-MnP-H4_FT-ChuA, SEQ ID NO:40) that is purified, cut with BsaI(NEB, Catalog No. R0535S), and ligated into the BfuA1-cut pJ1231-03Cvector using T4 DNA ligase (Life Technologies, Catalog No. 15224025) tocreate the pJ1231-AraC-MnP-H4_FT-ChuA expression construct. (Preferably,the BsaI-cut AraC-MnP-H4_FT-ChuA fragment is also ligated into theBfuA1-cut pJ1234-03C vector, to create the pJ1234-AraC-MnP-H4_FT-ChuAexpression construct.)

The pPRO33-PDI expression construct (SEQ ID NO:29), containing the PrpRcoding sequence, the pPRO propionate-inducible promoter, and the PDIcoding sequence, was used as the template in a PCR reaction, which addeda 5×His tag at the C-terminal end of the PDI coding sequence, and BsaIrestriction sites for cloning into the vectors. (Preferably, butoptionally, the PDI coding sequence is not altered to add a His tag atits C-terminal end; this can be accomplished by using a primer similarin sequence to that of SEQ ID NO:42, but without bases 21 through 35 ofSEQ ID NO:42.) The primers used in this PCR reaction were theBsaI-PrpR-PDI primer (SEQ ID NO:41) and the PDI-5×His-BsaI primer (SEQID NO:42), creating a PCR product (PrpR-PDI, SEQ ID NO:43) that waspurified, cut with BsaI, and ligated into the BfuA1-cut pJ1231-03Cvector using T4 DNA ligase to create the pJ1231-PrpR-PDI expressionconstruct. (Preferably, the BsaI-cut PrpR-PDI fragment is also ligatedinto the BfuA1-cut pJ1234-03C vector to create the pJ1234-PrpR-PDIexpression construct.)

The expression constructs encoding the mouse anti-human CD19 IgG1 heavychain and the mouse anti-human CD19 IgG1 light chain—pBAD24-HC andpPRO33-LC, respectively—were used as the templates in two separate PCRreactions each in order to remove the signal sequence from each codingsequence and to add BsaI restriction sites for cloning into the vectors.For pBAD24-HC, the primers used in these two PCR reactions were (1) theBsaI-AraC-HC-forward primer (SEQ ID NO:44) and the HC-reverse primer(SEQ ID NO:45), and (2) the HC-forward primer (SEQ ID NO:46) and theHC-BsaI-reverse primer (SEQ ID NO:47). For pPRO33-LC, the primers usedin these two PCR reactions were (1) the BsaI-PrpR-LC-forward primer (SEQID NO:48) and the LC-reverse primer (SEQ ID NO:49), and (2) theLC-forward primer (SEQ ID NO:50) and the LC-BsaI-reverse primer (SEQ IDNO:51). Use of the BsaI-AraC-HC-forward primer (SEQ ID NO:44) and theBsaI-PrpR-LC-forward primer (SEQ ID NO:48) in these reactions results inHC and LC coding sequences lacking the signal sequence, referred to asHC_NS (no signal) and LC_NS: the modified HC_NS coding sequence resultsin an HC_NS polypeptide having an initial methionine residue followed byamino acids 20 through 464 of SEQ ID NO:2; the modified LC_NS codingsequence results in an LC_NS polypeptide having an initial methionineresidue followed by an alanine residue and then amino acids 21 through239 of SEQ ID NO:4. A further PCR reaction was then performed on eachset of two purified PCR products, using the BsaI-AraC-HC-forward andHC-BsaI-reverse primers, and the BsaI-PrpR-LC-forward andLC-BsaI-reverse primers, respectively, to create two individual products(AraC-HC_NS, SEQ ID NO:52, and PrpR-LC_NS, SEQ ID NO:53) that were eachpurified, cut with BsaI, and ligated into the BfuA1-cut pJ1231-03Cvector (PrpR-LC_NS) or the BfuA1-cut pJ1234-03C vector (AraC-HC_NS)using T4 DNA ligase, to create the pJ1231-PrpR-LC_NS andpJ1234-AraC-HC_NS expression constructs.

The ligase mixtures were transformed into E. coli DH5alpha cells andplated on LB agar plates with sufficient amounts of kanamycin orampicillin to maintain the plasmids in the DH5alpha cells. Preparationof plasmid DNA was performed according to standard methods. (Optionallybut preferably, the prepared plasmid DNA is used in sequencing reactionsto confirm the sequences of the plasmid inserts.)

Competent INVSc-1 S. cerevisiae cells were prepared using the S.c.EasyComp™ Transformation Kit (Life Technologies, Catalog No. K5050-01)according to the manufacturer's instructions. The INVSc-1 S. cerevisiaestrain has the following genotype (MATa his3Δ1 leu2 trp1-289ura3-52/MATa his3Δ1 leu2 trp1-289 ura3-52) and phenotype: His⁻, Leu⁻,Trp⁻, Ura⁻. Briefly, a single colony from the INVSc-1 strain wasinoculated in 10 milliliters of YPD medium (contains, per liter: 10 gyeast extract, 20 g peptone, 20 g glucose) and grown overnight at 30degrees C. in a shaking incubator at 250 rpm. Next day, the overnightculture was diluted in 10 milliliters fresh YPD medium to an OD600 of0.3 and grown until OD600 reached 0.8. The cells were collected bycentrifugation at 1500 rpm for 5 minutes at room temperature. Afterthat, the cells were resuspended in 10 milliliters of Solution 1 (washsolution) and collected by centrifugation at 1500 rpm for 5 minutes atroom temperature. Supernatant was discarded and cells were resuspendedin 1 milliliter of Solution 2 (resuspension solution), divided in50-microliter aliquots and stored at −80 degrees C. To transform thecompetent yeast cells with the expression constructs, 50 microliters ofINVSc-1 competent cells were mixed with 1.2 micrograms of eachexpression vector or control vector and 500 microliters of Solution 3(transformation solution). Then cells were mixed vigorously andincubated in a 30 degrees C. water bath for 1 hour and vortexed for 10seconds every 15 minutes. 100- and 400-microliter aliquots oftransformation mixtures were seeded on SC minimal agar plates in theabsence of appropriate selective reagent. SC minimal medium contains,per liter: 6.7 g yeast nitrogen base, 20 g glucose, 0.05 g asparticacid, 0.05 g histidine, 0.05 g isoleucine, 0.05 g methionine, 0.05 gphenylalanine, 0.05 g proline, 0.05 g serine, 0.05 g tyrosine, 0.05 gvaline, 0.1 g adenine, 0.1 g arginine, 0.1 g cysteine, 0.1 g leucine(omitted in—Leu selective media), 0.1 g lysine, 0.1 g threonine, 0.1 gtryptophan, and 0.1 g uracil (omitted in—Ura selective media). INVSc-1cells transformed with pJ1231-PrpR-PDI, with pJ1231-PrpR-LC_NS, and withthe pJ1231-03C control vector were selected on plates without leucine at30 degrees C. for 48-72 hours. INVSc-1 cells transformed withpJ1234-AraC-HC_NS and with the pJ1234-03C control vector were grownunder the same conditions on SC minimal agar plates without uracil.(Preferably, INVSc-1 cells transformed with pJ1231-AraC-MnP-H4_FT-ChuA,with pJ1234-AraC-MnP-H4_FT-ChuA, and with pJ1234-PrpR-PDI are also grownunder the same conditions on SC minimal agar plates without uracil.)INVSc-1 cells co-transformed simultaneously with pJ1234-AraC-HC_NS andpJ1231-PrpR-LC_NS were selected under the same conditions on SC minimalagar plates without leucine or uracil. (Preferably, INVSc-1 cellsco-transformed simultaneously with pJ1231-AraC-MnP-H4_FT-ChuA andpJ1234-PrpR-PDI, or with pJ1234-AraC-MnP-H4_FT-ChuA and pJ1231-PrpR-PDI,are selected under the same conditions on SC minimal agar plates withoutleucine or uracil.)

Cells from all colonies from each transformation were scraped andresuspended in 4 milliliters of liquid minimal SC medium in absences ofappropriate selective reagent and carbon source. OD₆₀₀ in each culturewas measured and normalized to 0.4 optical units. Protein expression wasinduced by addition of 2% sterile filtered arabinose (Sigma-Aldrich, St.Louis, Mo., Catalog No. A3256-25G) to cultures transformed withpJ1234-AraC-HC_NS. In cells transformed with pJ1231-PrpR-PDI and withpJ1231-PrpR-LC_NS, protein expression was induced by addition of 2%sterile filtered propionate (Sigma-Aldrich, Catalog No. P188-100G). Andco-expression of pJ1234-AraC-HC_NS and pJ1231-PrpR-LC_NS incorresponding culture was induced by addition of 1% arabinose and 1%propionate. (Preferably, the induction medium for protein expression bycells transformed with pJ1231-AraC-MnP-H4_FT-ChuA, and for cellsco-transformed with pJ1231-AraC-MnP-H4_FT-ChuA and pJ1234-PrpR-PDI, orwith pJ1234-AraC-MnP-H4_FT-ChuA and pJ1231-PrpR-PDI, also contains hemin(Sigma-Aldrich, Catalog Nos. H9039 or 51280) added to a finalconcentration of 8 micromolar.) The time course for protein expressionwas 24 hours, at 30 degrees C. in a shaking incubator at 250 rpm. Cellsfrom 0.5 milliliters of each pre-induced cultures were collected bycentrifugation, washed with 1 milliliter of deionized water and storedat −80 degrees C. Samples from post-induced cultures were prepared inthe same way. Total protein extracts were prepared from pre- andpost-induced cultures, resolved in 4-20% SDS-PAGE, and transferred to aPDVF membrane. Expression level of target proteins was analyzed byWestern blotting using anti-6×His tag and anti-Human IgG antibodies.

Example 7

Titration of Coexpression by Varying Inducer Concentration

To optimize production of a multimeric product using the induciblecoexpression systems of the invention, it is possible to independentlyadjust or titrate the concentrations of the inducers. Host cellscontaining L-arabinose-inducible and propionate-inducible expressionconstructs are grown to the desired density (such as an OD₆₀₀ ofapproximately 0.5) in M9 minimal medium containing the appropriateantibiotics, then cells are aliquoted into small volumes of M9 minimalmedium, optionally prepared with no carbon source such as glycerol, andwith the appropriate antibiotics and varying concentrations of eachinducer. The concentration of L-arabinose necessary to induce expressionis typically less than 2%. In a titration experiment, the testedconcentrations of L-arabinose can range from 2% to 1.5%, 1%, 0.5%, 0.2%,0.1%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.005%, 0.002%, and 0.001%. Theconcentrations of L-rhamnose or D-xylose necessary to induce expressionof L-rhamnose-inducible or D-xylose-inducible promoters are testedsimilarly, with the tested concentrations ranging from 5% to 0.01%. Foreach concentration ‘x’ of L-arabinose (or L-rhamnose or D-xylose) thatis tested, the concentration of a different inducer such as propionate,added to each of the tubes containing concentration ‘x’ of the firstinducer, is varied in each series of samples, which in the case ofpropionate range from 1 M to 750 mM, 500 mM, 250 mM, 100 mM, 75 mM, 50mM, 25 mM, 10 mM, 5 mM, and 1 mM. Alternatively, titration experimentscan start at a ‘standard’ combination of inducer concentrations, whichis 0.002% of any of L-arabinose, L-rhamnose, or D-xylose, and/or 50 mMpropionate, and test new combinations of inducer concentrations thatvary from that of the ‘standard’ combination. Similar titrationexperiments can be performed with any combination of inducers used in aninducible coexpression system of the invention, including but notlimited to L-arabinose, propionate, L-rhamnose, and D-xylose. Aftergrowth in the presence of inducers for 6 hours, the cells are pelleted,the desired product is extracted from the cells, and the yield ofproduct per mass value of cells is determined by a quantitativeimmunological assay such as ELISA, or by purification of the product andquantification by UV absorbance at 280 nm.

It is also possible to titrate inducer concentrations using ahigh-throughput assay, in which the proteins to be expressed areengineered to include a fluorescent protein moiety, such as thatprovided by the mKate2 red fluorescent protein (Evrogen, Moscow,Russia), or the enhanced green fluorescent proteins from Aequoreavictoria and Bacillus cereus. Another approach to determining the amountand activity of gene products produced by different concentrations ofinducers in a high-throughput titration experiment, is to use a sensorcapable of measuring biomolecular binding interactions, such as a sensorthat detects surface plasmon resonance, or a sensor that employsbio-layer interferometry (BLI) (for example, an Octet® QK system fromforteBIO, Menlo Park, Calif.).

Example 8

Measurement of the Strength of Promoters and the Homogeneity ofInducible Expression

The strength of a promoter is measured as the amount of transcription ofa gene product initiated at that promoter, relative to a suitablecontrol. For constitutive promoters directing expression of a geneproduct in an expression construct, a suitable control could use thesame expression construct, except that the ‘wild-type’ version of thepromoter, or a promoter from a ‘housekeeping’ gene, is used in place ofthe promoter to be tested. For inducible promoters, expression of thegene product from the promoter can be compared under inducing andnon-inducing conditions.

A. Measuring Promoter Strength Using Quantitative PCR to DetermineLevels of RNA Transcribed from the Promoter

The method of De Mey et al. (“Promoter knock-in: a novel rational methodfor the fine tuning of genes”, BMC Biotechnol 2010 March 24; 10: 26) isused to determine the relative strength of promoters in host cells thatcan be grown in culture. Host cells containing an expression constructwith the promoter to be tested, and control host cells containing acontrol expression construct, are grown in culture in triplicate. One-mlsamples are collected at OD₆₀₀=1.0 for mRNA and protein collection.Total RNA extraction is done using an RNeasy mini kit (QIAGEN, TheNetherlands). The purity of RNA is verified on a FA-agarose gel asrecommended by QIAGEN and the RNA concentration is determined bymeasuring the absorbance at 260 nm. Two micrograms of RNA is used tosynthesize cDNA using a random primer and RevertAid H Minus M-MulVreverse transcriptase (Fermentas, Glen Burnie, Md.). The strength of thepromoter is determined by RT-qPCR carried out in an iCycler IQ®(Bio-Rad, Eke, Belgium) using forward and reverse primers designed toamplify the cDNA corresponding to the transcript produced from thepromoter. (For this purpose, the De Mey et al. authors used theFw-ppc-qPCR and Rv-ppc-qPCR primers, and the primers Fw-rpoB-qPCR andRv-rpoB-qPCR from the control housekeeping gene rpoB.) SYBR GreenER qPCRsupermix (Life Technologies, Grand Island, N.Y.) is used to perform abrief UDG (uracil DNA glycosylase) incubation (50° C. for 2 min)immediately followed by PCR amplification (95° C. for 8.5 min; 40 cyclesof 95° C. for 15 s and 60° C. for 1 min) and melting curve analysis (95°C. for 1 min, 55° C. for 1 min and 80 cycles of 55° C.+0.5° C./cyclesfor 10 s) to identify the presence of primer dimers and analyze thespecificity of the reaction. This UDG incubation step before PCR cyclingdestroys any contaminating dU-containing products from previousreactions. UDG is then inactivated by the high temperatures duringnormal PCR cycling, thereby allowing the amplification of genuine targetsequences. Each sample is performed in triplicate. The relativeexpression ratios are calculated using the “Delta-delta ct method” of PEApplied Biosystems (PerkinElmer, Forster City, Calif.).

B. Measuring Inducible Promoter Strength and Homogeneity of InductionUsing a Fluorescent Reporter Gene

These experiments are performed using the methods of Khlebnikov et al.(“Regulatable arabinose-inducible gene expression system with consistentcontrol in all cells of a culture”, J Bacteriol 2000 December; 182(24):7029-7034). Experiments measuring the induction of an inducible promoterare performed in C medium supplemented with 3.4% glycerol as a carbonsource (Helmstetter, “DNA synthesis during the division cycle of rapidlygrowing Escherichia coli B/r”, J Mol Biol 1968 Feb. 14; 31(3): 507-518).E. coli strains containing expression constructs comprising at least oneinducible promoter controlling expression of a fluorescent reporter geneare grown at 37° C. under antibiotic selection to an optical density at600 nm (OD600) of 0.6 to 0.8. Cells are collected by centrifugation(15,000×g), washed in C medium without a carbon source, resuspended infresh C medium containing antibiotics, glycerol, and/or inducer (for theinduction of gene expression) to an OD600 of 0.1 to 0.2, and incubatedfor 6 h. Samples are taken routinely during the growth period foranalysis. Culture fluorescence is measured on a Versafluor Fluorometer(Bio-Rad Inc., Hercules, Calif.) with 360/40-nm-wavelength excitationand 520/10-nm-wavelength emission filters. The strength of expressionfrom an inducible promoter upon induction can be expressed as the ratioof the maximum population-averaged fluorescence (fluorescence/OD ratio)of the induced cells relative to that of control (such as uninduced)cells. To determine the homogeneity of induction within the cellpopulation, flow cytometry is performed on a Beckman-Coulter EPICS XLflow cytometer (Beckman Instruments Inc., Palo Alto, Calif.) equippedwith an argon laser (emission at a wavelength of 488 nm and 15 mW) and a525-nm-wavelength band pass filter. Prior to the analysis, sampled cellsare washed with phosphate-buffered saline that had been filtered (filterpore size, 0.22 micrometers), diluted to an OD600 of 0.05, and placed onice. For each sample, 30,000 events are collected at a rate between 500and 1,000 events/s. The percentage of induced (fluorescent) cells ineach sample can be calculated from the flow cytometry data.

Example 9

Purification of Antibodies

Antibodies produced by the inducible coexpression systems of theinvention are purified by centrifuging samples of lysed host cells at10,000×g for 10 minutes to remove any cells and debris. The supernatantis filtered through a 0.45 micrometer filter. A 1-ml Recombinant ProteinG—Sepharose® 4B column (Life Technologies, Grand Island, N.Y.) is set upto achieve flow rates of 1 ml/min, and is used with the followingbuffers: binding buffer: 0.02 M sodium phosphate, pH 7.0; elutionbuffer: 0.1 M glycine-HCl, pH 2.7; and neutralization buffer: 1 MTris-HCl, pH 9.0. The column is equilibrated with 5 column volumes (5ml) of binding buffer, and then the sample is applied to the column. Thecolumn is washed with 5-10 column volumes of the binding buffer toremove impurities and unbound material, continuing until no protein isdetected in the eluent (determined by UV absorbance at 280 nm). Thecolumn is then eluted with 5 column volumes of elution buffer, and thecolumn is immediately re-equilibrated with 5-10 column volumes ofbinding buffer.

Example 10

Measurement of Antibody Binding Affinity

The antibody binding affinity, expressed as “Kd” or “Kd value”, ismeasured by a radiolabeled antigen-binding assay (RIA) performed withthe Fab version of an antibody of interest and its antigen as describedby the following assay. Production of the Fab version of a full-lengthantibody is well known in the art. Solution-binding affinity of Fabs forantigen is measured by equilibrating Fab with a minimal concentration of(¹²⁵I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (see, for example, Chen et al., “Selection andanalysis of an optimized anti-VEGF antibody: crystal structure of anaffinity-matured Fab in complex with antigen”, J Mol Biol 1999 Nov. 5;293(4): 865-881). To establish conditions for the assay, microtiterplates (DYNEX Technologies, Inc., Chantilly, Va.) are coated overnightwith 5 micrograms/ml of a capturing anti-Fab antibody (Cappel Labs, WestChester, Pa.) in 50 mM sodium carbonate (pH 9.6), and subsequentlyblocked with 2% (w/v) bovine serum albumin in PBS for two to five hoursat room temperature (approximately 23° C.). In a non-adsorbent plate(Nunc #269620; Thermo Scientific, Rochester, N.Y.), 100 μM or 26 μM[¹²⁵I]-antigen are mixed with serial dilutions of a Fab of interest(e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, inPresta et al.,“Humanization of an anti-vascular endothelial growthfactor monoclonal antibody for the therapy of solid tumors and otherdisorders”, Cancer Res 1997 Oct. 15; 57(20): 4593-4599). The Fab ofinterest is then incubated overnight; however, the incubation maycontinue for a longer period (e.g., about 65 hours) to ensure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature (e.g., for one hour).The solution is then removed and the plate washed eight times with 0.1%TWEEN-20™ surfactant in PBS. When the plates have dried, 150microliters/well of scintillant (MICROSCINT-20™; PerkinElmer, Waltham,Mass.) is added, and the plates are counted on a TOPCOUNT™ gamma counter(PerkinElmer) for ten minutes. Concentrations of each Fab that give lessthan or equal to 20% of maximal binding are chosen for use incompetitive-binding assays.

Alternatively, the Kd or Kd value is measured by using surface-plasmonresonance assays using a BIACORE®-2000 or a BIACORE®-3000 instrument(BIAcore, Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5chips at ^(˜)10 response units (RU). Briefly, carboxymethylated dextranbiosensor chips (CM5, BIAcore Inc.) are activated withN-ethyl-N′-(3-dimethylamino-propyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 micrograms/mlC0.2 micromolar) before injection at a flow rate of 5 microliters/minuteto achieve approximately 10 RU of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% TWEEN 20™ surfactant(PBST) at 25° C. at a flow rate of approximately 25 microliters/min.Association rates (k_(on)) and dissociation rates (k_(off)) arecalculated using a simple one-to-one Langmuir binding model (BIAcore®Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio k_(off)/k_(on). If the on-rateexceeds 10⁶ M⁻¹s⁻¹ by the surface-plasmon resonance assay above, thenthe on-rate can be determined by using a fluorescent quenching techniquethat measures the increase or decrease in fluorescence-emissionintensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25°C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in thepresence of increasing concentrations of antigen as measured in aspectrometer, such as a stop-flow-equipped spectrophotometer (AvivInstruments) or a 8000-series SLM-AMINCO™ spectrophotometer(ThermoSpectronic) with a stirred cuvette.

Example 11

Characterizing the Disulfide Bonds Present in Coexpression Products

The number and location of disulfide bonds in coexpressed proteinproducts can be determined by digestion of the protein with a protease,such as trypsin, under non-reducing conditions, and subjecting theresulting peptide fragments to mass spectrometry (MS) combiningsequential electron transfer dissociation (ETD) and collision-induceddissociation (CID) MS steps (MS2, MS3) (Nili et al., “Defining thedisulfide bonds of insulin-like growth factor-binding protein-5 bytandem mass spectrometry with electron transfer dissociation andcollision-induced dissociation”, J Biol Chem 2012 Jan. 6; 287(2):1510-1519; Epub 2011 Nov. 22).

Digestion of Coexpressed Protein.

To prevent disulfide bond rearrangements, any free cysteine residues arefirst blocked by alkylation: the coexpressed protein is incubatedprotected from light with the alkylating agent iodoacetamide (5 mM) withshaking for 30 minutes at 20° C. in buffer with 4 M urea, and then isseparated by non-reducing SDS-PAGE using precast gels. Alternatively,the coexpressed protein is incubated in the gel after electrophoresiswith iodoacetamide, or without as a control. Protein bands are stained,de-stained with double-deionized water, excised, and incubated twice in500 microliters of 50 mM ammonium bicarbonate, 50% (v/v) acetonitrilewhile shaking for 30 minutes at 20° C. Protein samples are dehydrated in100% acetonitrile for 2 minutes, dried by vacuum centrifugation, andrehydrated with 10 mg/ml of trypsin or chymotrypsin in buffer containing50 mM ammonium bicarbonate and 5 mM calcium chloride for 15 minutes onice. Excess buffer is removed and replaced with 50 microliters of thesame buffer without enzyme, followed by incubation for 16 hours at 37°C. or 20° C., for trypsin and chymotrypsin, respectively, with shaking.Digestions are stopped by addition of 3 microliters of 88% formic acid,and after brief vortexing, the supernatant is removed and stored at −20°C. until analysis.

Localization of Disulfide Bonds by Mass Spectrometry.

Peptides are injected onto a 1 mm×8 mm trap column (MichromBioResources, Inc., Auburn, Calif.) at 20 microliters/minute in a mobilephase containing 0.1% formic acid. The trap cartridge is then placedin-line with a 0.5 mm×250 mm column containing 5 mm Zorbax SB-C18stationary phase (Agilent Technologies, Santa Clara, Calif.), andpeptides separated by a 2-30% acetonitrile gradient over 90 minutes at10 micro-liters/minute with a 1100 series capillary HPLC (AgilentTechnologies). Peptides are analyzed using a LTQ Velos linear ion trapwith an ETD source (Thermo Scientific, San Jose, Calif.). Electrosprayionization is performed using a Captive Spray source (MichromBioresources, Inc.). Survey MS scans are followed by sevendata-dependant scans consisting of CID and ETD MS2 scans on the mostintense ion in the survey scan, followed by five MS3 CID scans on thefirst- to fifth-most intense ions in the ETD MS2 scan. CID scans usenormalized collision energy of 35, and ETD scans use a 100 insactivation time with supplemental activation enabled. Minimum signals toinitiate MS2 CID and ETD scans are 10,000, minimum signals forinitiation of MS3 CID scans are 1000, and isolation widths for all MS2and MS3 scans are 3.0 m/z. The dynamic exclusion feature of the softwareis enabled with a repeat count of 1, exclusion list size of 100, andexclusion duration of 30 s. Inclusion lists to target specificcross-linked species for collection of ETD MS2 scans are used. Separatedata files for MS2 and MS3 scans are created by Bioworks 3.3 (ThermoScientific) using ZSA charge state analysis. Matching of MS2 and MS3scans to peptide sequences is performed by Sequest (V27, Rev 12, ThermoScientific). The analysis is performed without enzyme specificity, aparent ion mass tolerance of 2.5, fragment mass tolerance of 1.0, and avariable mass of +16 for oxidized methionine residues. Results are thenanalyzed using the program Scaffold (V3_00_08, Proteome Software,Portland, Oreg.) with minimum peptide and protein probabilities of 95and 99% being used. Peptides from MS3 results are sorted by scan number,and cysteine containing peptides are identified from groups of MS3 scansproduced from the five most intense ions observed in ETD MS2 scans. Theidentities of cysteine peptides participating in disulfide-linkedspecies are further confirmed by manual examination of the parent ionmasses observed in the survey scan and the ETD MS2 scan.

Example 12

Isolation of Coexpression Products from Bacterial Cell Periplasm, fromSpheroplasts, and from Whole Cells

The inducible coexpression system of the invention can be used toexpress gene products that accumulate in different compartments of thecell, such as the cytoplasm or periplasm. Host cells such as E. coli orS. cerevisiae have an outer cell membrane or cell wall, and can formspheroplasts when the outer membrane or wall is removed. Coexpressedproteins made in such hosts can be purified specifically from theperiplasm, or from spheroplasts, or from whole cells, using thefollowing method (Schoenfeld, “Convenient, rapid enrichment ofperiplasmic and spheroplasmic protein fractions using the new PeriPreps™Periplasting Kit”, Epicentre Forum 1998 5(1): 5; seewww.epibio.com/newsletter/f5_1/f5_1pp.asp). This method, using thePeriPreps™ Periplasting Kit (Epicentre® Biotechnologies, Madison Wis.;protocol available at wvvw.epibio.com/pdftechlit/107p10612.pdf), isdesigned for E. coli and other grain negative bacteria, but the generalapproach can be modified for other host cells such as S. cerevisiae.

1. The bacterial host cell culture is grown to late log phase only, asolder cell cultures in stationary phase commonly demonstrate someresistance to lysozyme treatment. If the expression of recombinantprotein is excessive, cells may prematurely lyse; therefore, cellcultures are not grown in rich medium or at higher growth temperaturesthat might induce excessive protein synthesis. Protein expression isthen induced; the cells should be in log phase or early stationaryphase.

2. The cell culture is pelleted by centrifugation at a minimum of1,000×g for 10 minutes at room temperature. Note: the cells must befresh, not frozen. The wet weight of the cell pellet is determined inorder to calculate the amount of reagents required for this protocol.

3. The cells are thoroughly resuspended in a minimum of 2 ml ofPeriPreps Periplasting Buffer (200 mM Tris-HCl pH 7.5, 20% sucrose, 1 mMEDTA, and 30 U/microliter Ready-Lyse Lysozyme) for each gram of cells,either by vortex mixing or by pipeting until the cell suspension ishomogeneous. Note: excessive agitation may cause premature lysing of thespheroplasts resulting in contamination of the periplasmic fraction withcytoplasmic proteins.

4. Incubate for five minutes at room temperature. Ready-Lyse Lysozyme isoptimally active at room temperature. Lysis at lower temperatures (0°C.-4° C.) requires additional incubation time; at such temperaturesincubation times are extended 2- to 4-fold.

5. Add 3 ml of purified water at 4° C. for each gram of original cellpellet weight (Step 2) and mix by inversion.

6. Incubate for 10 minutes on ice.

7. The lysed cells are pelleted by centrifugation at a minimum of4,000×g for 15 minutes at room temperature.

8. The supernatant containing the periplasmic fraction is transferred toa clean tube.

9. To degrade contaminating nucleic acids, OmniCleave Endonuclease isoptionally added to PeriPreps Lysis Buffer. Inclusion of a nuclease willgenerally improve the yield of protein and the ease of handling of thelysates, but addition of a nuclease is undesirable in some cases: forexample, the use of a nuclease should be avoided if residual nucleaseactivity or transient exposure to the magnesium cofactor will interferewith subsequent assays or uses of the purified protein. The addition ofEDTA to the lysate to inactivate OmniCleave Endonuclease, likewise, mayinterfere with subsequent assay or use of the purified protein. Ifnuclease is to be added, 2 microliters of OmniCleave Endonuclease and 10microliters of 1.0 M MgCl₂ are diluted up to 1 ml with PeriPreps LysisBuffer (10 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM EDTA, and 0.1%deoxycholate) for each milliliter of Lysis Buffer needed in Step 10.

10. The pellet is resuspended in 5 ml of PeriPreps Lysis Buffer for eachgrain of original cell pellet weight.

11. The pellet is incubated at room temperature for 10 minutes (ifincluded, OmniCleave Endonuclease activity will cause a significantdecrease in viscosity; the incubation is continued until the cellularsuspension has the consistency of water).

12. The cellular debris is pelleted by centrifugation at a minimum of4,000×g for 15 minutes at 4° C.

13. The supernatant containing the spheroplast fraction is transferredto a clean tube.

14. If OmniCleave Endonuclease was added to the PeriPreps Lysis Buffer,20 microliters of 500 mM EDTA is added for each milliliter of theresultant spheroplastic fraction, to chelate the magnesium (the finalconcentration of EDTA in the lysate is 10 mM). Following hydrolysis ofnucleic acids with OmniCleave Endonuclease, lysates may containsubstantial amounts of mono- or oligonucleotides. The presence of thesedegradation products may affect further processing of the lysate: forexample, nucleotides may decrease the binding capacity of anion exchangeresins by interacting with the resin.

The above protocol can be used to prepare total cellular protein withthe following modifications. The cells pelleted in Step 2 can be freshor frozen; at Step 4, the cells are incubated for 15 minutes; Steps 5through 8 are omitted; at Step 10, 3 ml of PeriPreps Lysis Buffer isadded for each gram of original cell pellet weight.

After preparation of periplasmic, or spheroplastic, or whole-cellprotein samples, the samples can be analyzed by any of a number ofprotein characterization and/or quantification methods. In one example,the successful fractionation of periplasmic and spheroplastic proteinsis confirmed by analyzing an aliquot of both the periplasmic andspheroplastic fractions by SDS-PAGE (two microliters of each fraction isgenerally sufficient for visualization by staining with CoomassieBrilliant Blue). The presence of unique proteins or the enrichment ofspecific proteins in a given fraction indicates successfulfractionation. For example, if the host cell contains a high-copy numberplasmid with the ampicillin resistance marker, then the presence ofβ-lactamase (31.5 kDa) mainly in the periplasmic fraction indicatessuccessful fractionation. Other E. coli proteins found in theperiplasmic space include alkaline phosphatase (50 kDa) and elongationfactor Tu (43 kDa). The amount of protein found in a given fraction canbe quantified using any of a number of methods (such as SDS-PAGE anddensitometry analysis of stained or labeled protein bands, scintillationcounting of radiolabeled proteins, enzyme-linked immunosorbent assay(ELISA), or scintillation proximity assay, among other methods.)Comparing the amounts of a protein found in the periplasmic fraction ascompared to the spheroplastic fraction indicates the degree to which theprotein has been exported from the cytoplasm into the periplasm.

Example 13

Determination of Polynucleotide or Amino Acid Sequence Similarity

Percent polynucleotide sequence or amino acid sequence identity isdefined as the number of aligned symbols, i.e. nucleotides or aminoacids, that are identical in both aligned sequences, divided by thetotal number of symbols in the alignment of the two sequences, includinggaps. The degree of similarity (percent identity) between two sequencesmay be determined by aligning the sequences using the global alignmentmethod of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), asimplemented by the National Center for Biotechnology Information (NCBI)in the Needleman-Wunsch Global Sequence Alignment Tool, availablethrough the website blast.ncbi.nlm.nih.gov/Blast.cgi. In one embodiment,the Needleman and Wunsch alignment parameters are set to the defaultvalues (Match/Mismatch Scores of 2 and -3, respectively, and Gap Costsfor Existence and Extension of 5 and 2, respectively). Other programsused by those skilled in the art of sequence comparison may also be usedto align sequences, such as, for example, the basic local alignmentsearch tool or BLAST® program (Altschul et al., “Basic local alignmentsearch tool”, J Mol Biol 1990 Oct. 5; 215(3): 403-410), as implementedby NCBI, using the default parameter settings described at theblast.ncbi.nlm nih.gov/Blast.cgi website. The BLAST algorithm hasmultiple optional parameters including two that may be used as follows:(A) inclusion of a filter to mask segments of the query sequence thathave low compositional complexity or segments consisting ofshort-periodicity internal repeats, which is preferably not utilized orset to ‘off’, and (B) a statistical significance threshold for reportingmatches against database sequences, called the ‘Expect’ or E-score (theexpected probability of matches being found merely by chance; if thestatistical significance ascribed to a match is greater than thisE-score threshold, the match will not be reported). If this ‘Expect’ orE-score value is adjusted from the default value (10), preferredthreshold values are 0.5, or in order of increasing preference, 0.25,0.1, 0.05, 0.01, 0.001, 0.0001, 0.00001, and 0.000001.

In practicing the present invention, many conventional techniques inmolecular biology, microbiology, and recombinant DNA technology areoptionally used. Such conventional techniques relate to vectors, hostcells, and recombinant methods. These techniques are well known and areexplained in, for example, Berger and Kimmel, Guide to Molecular CloningTechniques, Methods in Enzymology volume 152 Academic Press, Mc, SanDiego, Calif.; Sambrook et al., Molecular Cloning—A Laboratory Manual(3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 2000; and Current Protocols in Molecular Biology, F. M. Ausubel etal., eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (supplemented through2006). Other useful references, for example for cell isolation andculture and for subsequent nucleic acid or protein isolation, includeFreshney (1994) Culture of Animal Cells, a Manual of Basic Technique,third edition, Wiley-Liss, New York and the references cited therein;Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems JohnWiley & Sons, Inc. New York, N.Y.; Gamborg and Phillips (Eds.) (1995)Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer LabManual, Springer-Verlag (Berlin Heidelberg N.Y.); and Atlas and Parks(Eds.) The Handbook of Microbiological Media (1993) CRC Press, BocaRaton, Fla. Methods of making nucleic acids (for example, by in vitroamplification, purification from cells, or chemical synthesis), methodsfor manipulating nucleic acids (for example, by site-directedmutagenesis, restriction enzyme digestion, ligation, etc.), and variousvectors, cell lines, and the like useful in manipulating and makingnucleic acids are described in the above references. In addition,essentially any polynucleotide (including labeled or biotinylatedpolynucleotides) can be custom or standard ordered from any of a varietyof commercial sources.

The present invention has been described in terms of particularembodiments found or proposed to comprise certain modes for the practiceof the invention. It will be appreciated by those of ordinary skill inthe art that, in light of the present disclosure, numerous modificationsand changes can be made in the particular embodiments exemplifiedwithout departing from the intended scope of the invention.

All cited references, including patent publications, are incorporatedherein by reference in their entirety. Nucleotide and other geneticsequences, referred to by published genomic location or otherdescription, are also expressly incorporated herein by reference.

SEQUENCES PRESENTED IN THE SEQUENCE LISTING SEQ ID NO: Length: Type:Organism: Description; ‘Other Information’ 1 1526 DNA Mus musculusCoding sequence for the mouse anti- human CD19 IgG1 heavy chain 2 464PRT Mus musculus Full-length mouse anti-human CD19 IgG1 heavy chainamino acid sequence 3 958 DNA Mus musculus Coding sequence for the mouseanti- human CD19 IgG1 light chain 4 239 PRT Mus musculus Full-lengthmouse anti-human CD19 IgG1 light chain amino acid sequence 5 1429 DNAArtificial Sequence Optimized coding sequence for the mouse anti-humanCD19 IgG1 heavy chain 6 757 DNA Artificial Sequence Optimized codingsequence for the mouse anti-human CD19 IgG1 light chain 7 5874 DNAArtificial Sequence pPRO33 vector 8 36 DNA Artificial Sequence IgG1HC Fcforward primer 9 32 DNA Artificial Sequence IgG1HC Fc reverse primer 1019 DNA Artificial Sequence pBAD24/33 forward primer 11 17 DNA ArtificialSequence pBAD24/33 reverse primer #1 12 17 DNA Artificial SequencepBAD24/33 reverse primer #2 13 370 PRT Artificial Sequence P.chrysosporium MnP-H4 amino acid sequence, without signal peptide 14 1113DNA Artificial Sequence Optimized coding sequence for P. chrysosporiumMnP-H4, without signal peptide 15 370 PRT Artificial Sequence P.chrysosporium MnP-H4 A60C/A75C amino acid sequence, without signalpeptide 16 1113 DNA Artificial Sequence Optimized coding sequence for P.chrysosporium MnP-H4 A60C/A75C, without signal peptide 17 660 PRT E.coli O157:H7 E. coli O157:H7 str. EC4113 ChuA amino acid sequence 181276 DNA Artificial Sequence MnP-H4 expression construct 19 2056 DNAArtificial Sequence ChuA expression construct 20 3326 DNA ArtificialSequence MnP-H4 ChuA expression construct 21 486 PRT Artificial SequenceHumicola insolens PDI amino acid sequence, without signal peptide 221487 DNA Artificial Sequence PDI expression construct 23 359 PRTArtificial Sequence P. chrysosporium MnP-H4 amino acid sequence, matureand “fully truncated” 24 25 DNA Artificial Sequence MnP-H4_FT NcoIforward primer 25 27 DNA Artificial Sequence MnP-H4_FT SalI reverseprimer 26 24 DNA Artificial Sequence ChuA SalI forward primer 27 31 DNAArtificial Sequence ChuA HindIII reverse primer 28 7768 DNA ArtificialSequence pBAD24-MnP_FT-ChuA expression construct 29 7316 DNA ArtificialSequence pPRO33-PDI expression construct 30 451 PRT Artificial SequenceInfliximab chimeric (murine variable doman, human constant domain) heavychain 31 215 PRT Artificial Sequence Infliximab chimeric (murinevariable doman, human constant domain) light chain 32 5875 DNAArtificial Sequence pBAD24-Infliximab_HC expression construct 33 6503DNA Artificial Sequence pPRO33-Infliximab_LC expression construct 346545 DNA Artificial Sequence pJ1231-03C plasmid 35 6009 DNA ArtificialSequence pJ1234-03C plasmid 36 42 DNA Artificial SequenceBsaI-AraC-MnP-H4_FT primer 37 63 DNA Artificial SequenceMnP-H4_FT-6xHis-reverse primer 38 38 DNA Artificial SequenceMnP-H4_FT-6xHis-forward primer 39 66 DNA Artificial SequenceChuA-6xHis-BsaI primer 40 4569 DNA Artificial SequenceAraC-MnP-H4_FT-ChuA PCR product 41 42 DNA Artificial SequenceBsaI-PrpR-PDI primer 42 55 DNA Artificial Sequence PDI-5xHis-BsaI primer43 3326 DNA Artificial Sequence PrpR-PDI PCR product 44 44 DNAArtificial Sequence BsaI-AraC-HC-forward primer 45 46 DNA ArtificialSequence HC-reverse primer 46 46 DNA Artificial Sequence HC-forwardprimer 47 45 DNA Artificial Sequence HC-BsaI-reverse primer 48 43 DNAArtificial Sequence BsaI-PrpR-LC-forward primer 49 50 DNA ArtificialSequence LC-reverse primer 50 50 DNA Artificial Sequence LC-forwardprimer 51 43 DNA Artificial Sequence LC-BsaI-reverse primer 52 2615 DNAArtificial Sequence AraC-HC_NS PCR product 53 2584 DNA ArtificialSequence PrpR-LC_NS PCR product

What is claimed is: 1-57. (canceled)
 58. A host cell suitable forexpression of at least one gene product from two or more induciblepromoters, wherein at least one of said inducible promoters isresponsive to an inducer that is different than the inducer of anotherof said inducible promoters, and each inducible promoter is selectedfrom the group consisting of: an L-arabinose-inducible promoter, apropionate-inducible promoter, a rhamnose-inducible promoter, and aD-xylose-inducible promoter; wherein the host cell has an altered genefunction of at least one gene that affects the reduction/oxidationenvironment of the host cell cytoplasm; and wherein the host cell has areduced level of gene function of at least one gene encoding a proteinthat metabolizes a first inducer of at least a first one of saidinducible promoters; and wherein the host cell has a reduced level ofgene function of at least one gene encoding a protein involved inbiosynthesis of a second inducer of at least a second one of saidinducible promoters, wherein the second inducer is different than thesaid first inducer.
 59. The host cell of claim 58 wherein the host cellis a prokaryotic cell.
 60. The host cell of claim 59 wherein the hostcell is an E. coli cell.
 61. The host cell of claim 60 wherein at leastone inducible promoter is selected from the group consisting of: thearaBAD promoter, the prpBCDE promoter, the rhaSR promoter, and the xlyApromoter.
 62. The host cell of claim 60 wherein at least one gene thataffects the reduction/oxidation environment of the host cell cytoplasmis trxB.
 63. The host cell of claim 58 wherein the host cell expresses amutant form of AhpC.
 64. The host cell of claim 60 wherein at least onegene encoding a protein that metabolizes a first inducer of at least afirst one of said inducible promoters is selected from the groupconsisting of araA, araB, prpB, prpD, rhaA, rhaB, rhaD, xylA, and xylB.65. The host cell of claim 60 wherein at least one gene encoding aprotein involved in biosynthesis of a second inducer of at least asecond one of said inducible promoters is selected from the groupconsisting of scpA/sbm, argK/ygfD, scpB/ygfG, scpC/ygfH, rmlA, rmlB,rmlC, and rmlD.
 66. The host cell of claim 58 wherein the host cell hasan alteration of gene function of at least one gene encoding atransporter protein for an inducer of at least one said induciblepromoter.
 67. The host cell of claim 60 wherein the host cell has analteration of gene function of at least one gene encoding a transporterprotein for an inducer of at least one said inducible promoter, whereinsaid gene encoding a transporter protein is selected from the groupconsisting of araE, araF, araG, araH, rhaT, xylF, xylG, and xylH. 68.The host cell of claim 67 wherein the gene encoding a transporterprotein is araE, and the alteration of gene function of said araE geneis changing the promoter controlling expression of the host cell'schromosomal araE gene from an arabinose-inducible promoter to aconstitutive promoter.
 69. The host cell of claim 58 wherein the hostcell comprises at least one polynucleotide encoding a form of DsbClacking a signal peptide.
 70. The host cell of claim 58 furthercomprising at least one expression construct comprising at least oneinducible promoter and a polynucleotide sequence encoding at least onegene product to be transcribed from the inducible promoter.
 71. The hostcell of claim 70 wherein at least one gene product is a polypeptide thatlacks a signal peptide.
 72. The host cell of claim 71 wherein at leastone gene product forms at least three disulfide bonds.
 73. A method ofproducing a gene product, the method comprising growing a culture of thehost cell of claim 70 and adding an inducer of at least one induciblepromoter to the culture.
 74. A host cell suitable for expression of atleast one gene product from two or more inducible promoters, wherein atleast one of said inducible promoters is responsive to an inducer thatis different than the inducer of another of said inducible promoters,and each inducible promoter is selected from the group consisting of: anL-arabinose-inducible promoter, a propionate-inducible promoter, arhamnose-inducible promoter, and a D-xylose-inducible promoter; whereinthe host cell has an altered gene function of at least one gene thataffects the reduction/oxidation environment of the host cell cytoplasm;and wherein the host cell has a reduced level of gene function of atleast one gene encoding a protein that metabolizes a first inducer of atleast a first one of said inducible promoters; and wherein the host cellhas a reduced level of gene function of at least one gene encoding aprotein that metabolizes a second inducer of at least a second one ofsaid inducible promoters, wherein the second inducer is different thanthe said first inducer.
 75. The host cell of claim 74 wherein the hostcell is an E. coli cell.
 76. The host cell of claim 75 wherein at leastone inducible promoter is selected from the group consisting of: thearaBAD promoter, the prpBCDE promoter, the rhaSR promoter, and the xlyApromoter.
 77. The host cell of claim 75 wherein at least one gene thataffects the reduction/oxidation environment of the host cell cytoplasmis trxB.
 78. The host cell of claim 75 wherein at least one geneencoding a protein that metabolizes a first inducer of at least a firstone of said inducible promoters is selected from the group consisting ofaraA, araB, prpB, prpD, rhaA, rhaB, rhaD, xylA, and xylB.
 79. The hostcell of claim 74 wherein the host cell has a reduced level of genefunction of at least one gene encoding a protein involved inbiosynthesis of an inducer of at least one said inducible promoter. 80.The host cell of claim 74 further comprising at least one expressionconstruct comprising at least one inducible promoter and apolynucleotide sequence encoding at least one gene product to betranscribed from the inducible promoter.
 81. A method of producing agene product, the method comprising growing a culture of the host cellof claim 80 and adding an inducer of at least one inducible promoter tothe culture.